chimeric antigen receptors specific for b cell maturation antigens (bcma)

专利摘要:
The present invention relates to chimeric antigen receptors (CARs) that comprise an extracellular BCMA binding domain, in particular an scFv. The CAR also comprises a spacer of at least 125 amino acids in length, a trans-membrane domain, and a signaling region. It can likewise include an intracellular costimulatory domain. Likewise, genetically engineered cells that express CARs and their uses are provided as in adoptive cell therapy.
公开号:BR112020008638A2
申请号:R112020008638-0
申请日:2018-11-01
公开日:2020-10-20
发明作者:Blythe D. SATHER；Audrey Olshefsky；Stefan Ponko；Ruth SALMON；Semih Tareen；Rebecca Wu；Yan Chen；Csaba Pazmany；Steven M. Shamah；Mariana Cota Stirner；Jui DUTTA-SIMMONS；Eric L. Smith；Melissa WORKS；Rupesh Amin；Aye Chen；Kimberly Harrington；Collin HAUSKINS；Erik Hess；Cyr De Imus；Jon Jones
申请人:Juno Therapeutics Inc；Memorial Sloan Kettering Cancer Center；
IPC主号:

专利说明:

[001] [001] This application claims priority of provisional application US 62 / 580,439, filed on November 1, 2017, entitled "CHI-MERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION TENANT AND ENCODING POLYNUCLEOTIDES," provisional application US No 62 / 580,445, filed on November 10, 2017, entitled "CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B- CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTI-DES," provisional application No. 62 / 582,932, filed on November 7, 2017, entitled "CHIMERIC ANTIGEN RECEPTORS SPECI- FIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES," provisional application No. 62 / 582,938, deposited on November 7, 2017, entitled "CHIMERIC ANTIGEN RE- CEPTORS SPECIFIC FOR B -CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES, "US provisional application No. 62 / 596,765, filed on December 8, 2017, entitled" CHIME- RIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES, "US provisional application No. 62 / 596,763, filed on December 8, 2017, entitled "CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATU- RATION ANTIGEN AND ENCODING POLYNUCLEOTIDES," provisional application No. 62 / 614,960, filed on January 8, 2018, entitled "CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES," provisional application No. 62 / 614.963, filed on January 8, 2018, entitled "CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN ANDC POLYNUCLEO-
[002] [002] The present application is being submitted together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file titled 735042009940SeqList.txt, created on November 1, 2018, which is 593 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. Field
[003] [003] The present description refers in some respects to chimeric antigen receptors (CARs), which contain portions of antibodies specific for B cell maturation antigen (BCMA) and polynucleotides that encode specific CARs for BCMA . The description also refers to genetically modified cells, containing these BCMA-binding receptors, and their uses in adoptive cell therapy. Background
[004] [004] B cell maturation antigen (BCMA) is a type 11l transmembrane protein expressed in mature B lymphocytes. After BCMA binds to its ligands, TNF B cell activator (BAFF) or a proliferation-inducing ligand (APRIL), a pro-survival cell signal is released to the B cell that has been deemed necessary for survival plasma cells. The expression of
[005] [005] Polynucleotides encoding a chimeric antigen receptor are provided, containing nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, in which after expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide, exhibits at least 70%, 75%, 80% , 85%, 90% or 95% RNA homogeneity. In some cases, the spacer is derived from an immunoglobulin. In some modes, the spacer includes a sequence of a region of articulation, a region of Chn2 and Chx3. In some modalities, one of the most CH2 and Cx3 joints is derived wholly or partially from I9G4 or IgG2. In some cases, the Cr2 and Cn; 3 joint is derived from IgG4. In some aspects, one or more of the Ch2 and ChH3 joints are chimeric and contain sequences derived from IgG4 and IgG2. In some examples, the spacer contains an I9G4 / 2 chimeric joint, an IgG2 / 4 Ch2 region and an IgG4 Cx43 region. In some modalities, the encoded spacer is or contains (i) the sequence mentioned in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% of sequence identity for SEQ ID NO: 649; or (iii) a contiguous portion of (i) or (ii) with at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes the sequence mentioned in SEQ ID NO: 649.
[006] [006] In some modalities, the spacer has a length of 125 to 300 amino acids, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length, or 250 to 300 amino acids. In some embodiments, the spacer is at least or at least approximately or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225 , 226, 227, 228 or 229 amino acids in length, or a length between any of the above.
[007] [007] In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the spacer includes at least one modified binding donor and / or binding receptor site, said modified binding donor and / or receiving site binding site containing one or more nucleotide modifications corresponding to a reference binding donor site and / or reference binding receptor site contained in the sequence mentioned in SEQ ID
[008] [008] In some embodiments of any of the polynucleotides described herein, the reference donor binding site includes the sequence aatctaagtacggac (SEQ ID NO: 705), teaactggtacgtag (SEQ ID NO: 706), acaattagtaaggea (SEQ ID NO: 707) and / or accacaggtgatatt SEQ ID NO: 708); and / or the reference binding receptor site includes the sequence aagtttctttetatattecaggctgacegtagataaatctce (SEQ ID NO: 742) and / or gggcaacgtattctettgcagtgteatacacgaagecetge (SEQ ID NO: 743). In some embodiments, the reference binding donor site (s) and / or reference binding recipient (s) have a binding site prediction score of at least or about 0.70, 0 , 75, 0.80, 0.85, 0.90, 0.95, 0.99 or 1.0; and / or the referral donor site (s) and / or referral link recipient (s) is (are) expected to be involved in an event connection with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the donor site of the reference link contains the sequence tcaactagtacgtag (SEQ ID NO: 706); and / or the reference binding receptor site contains the following
[009] [009] In some embodiments of any of the polynucleotides described herein, at least one of the one or more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 junction residues of the binding site of the reference binding recipient site and / or reference binding donor. In some respects, the one or more nucleotide modifications are silent and / or result in a degenerate codon compared to SEQ ID NO: 621 and / or do not alter the amino acid sequence of the encoded spacer. In some embodiments, the modified binding donor site is mentioned in agtctaaatacggac (SEQ ID NO: 661), teaactagtatatag (SEQ ID NO: 662), accatctecaaggec (SEQ ID NO: 663) and / or gececagatttacac (SEQ ID NO: 663) NO: 664); and / or the modified binding receptor site is mentioned in cagtttcttcctatatagtagactcacegtagataaatecaa (SEQ ID NO: 672) gaggcaacgtattcagctgcagegtgatgcacgaggeceetge (SEQ ID NO: 673) and / or aagttttttttttttttttttttttttttttatt In some cases, the modified donor binding site is mentioned in tcaactagtatatag (SEQ ID NO: 662) and / or the modified recipient site is mentioned in cadgtttcttectatatagtagactcacegtggataaatecaa (SEQ ID NO: 672). In some of these embodiments, the spacer is encoded by a nucleotide sequence mentioned in SEQ ID NO: 622 or a portion thereof.
[0010] [0010] A polynucleotide encoding a chimeric antigen receptor is provided, wherein the polynucleotide includes nucleic acid encoding: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is or includes the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[0011] [0011] A polynucleotide encoding a chimeric antigen receptor is also provided, wherein the polynucleotide including the nucleic acid encoding: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid includes or mainly includes the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[0012] [0012] In some of the modalities, after the expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some modalities, after expression in a cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide exhibits reduced heterogeneity in comparison to the heterogeneity of the MRNA transcribed from a reference polynucleotide, the referred poly- reference nucleotide encoding the same amino acid sequence as the polynucleotide, where the reference polynucleotide differs in the presence of one or more binding donor sites and / or one or more binding receptor sites in the nucleic acid encoding the spacer and / or includes one or more nucleotide modifications compared to the polynucleotide. In some cases, the RNA heterogeneity is reduced by more or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the RNA transcribed, optionally, the messenger RNA (mMRNA), from the reference polynucleotide exhibits more or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50 % or more of RNA heterogeneity. In some of these modalities, RNA homogeneity and / or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation or liquid chromatography. In some of these modalities, the polynucleotide is codon-optimized.
[0013] [0013] In some modalities of any of the polynucleotides described here, the antigen is associated with the disease or condition or expressed in cells in the environment of a lesion associated with the disease or condition. In some cases, the disease or condition is cancer. In some instances, the disease or condition is a myeloma, leukemia or lymphoma. In some modalities, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin , CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa 2, erb -B2, erb-B3, erb-B4, erbB dimers, EGFR vill, folate-binding protein (FBP), FCRL5, FCRHS5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- alpha, IL-13R-alpha2, kinase insertion domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule, (LI-CAM), melanoma-associated antigen (MAGE) -A1, MAGE-A3, MAGE-AS6, preferentially expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, 11-13 alpha-13 receptor (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD4 4v 7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, fetal AchR, NKG2D ligands, CD44v6, dual antigen, cancer testis antigen, mesothelin, murine CMV, mucin 1 ( MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor, receptor for progesterone, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7,
[0014] [0014] In some of these modalities, the antigen-binding domain is an antibody fragment that contains a variable heavy chain (Vn4) and variable light chain (V.) region. In some respects, the Vu region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vn region amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609 617, 772-774, or 814-832; and / or the V region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534- 550, 552-557, 610,618, 775-777, or 833-849. In some cases, the Vn region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identi - sequence sequence for the Vn region amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and / or the V region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610,618, 775-777, or 833-849.
[0015] [0015] In some modalities of any of the polynucleotides described here, the Vx region is or contains a CDR-H1, CDR-
[0016] [0016] In some modalities of any of the polynucleotides described herein, the Vu region is or contains (a) a heavy chain 1 complementarity determining region (CDR-H1) containing the selected amino acid sequence from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) containing the selected amino acid sequence from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290 , 291, 372, 513, 594, 605 or 612; and (c) a heavy chain 3 complementarity determining region (CDR-H3) containing the selected amino acid sequence from any of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155 , 156, 157, 292, 293, 376, 517, 595, 606 or 613; and / or the V region is or contains (a) a light chain 1 complementarity determining region (CDR-L1) containing the selected amino acid sequence from any of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain 2 complementarity determination region (CDR-L2) containing the selected amino acid sequence from any of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain 3 complementarity determining region (CDR-L3) containing the selected amino acid sequence from any of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185 , 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603.
[0017] [0017] In some modalities of any of the polynucleotides described here, the Vx region contains a CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 1, 4, and 7, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 10, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 11, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 140, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 141, 145, and 150, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 142, 146, and 151, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 152, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 143, 147, and 153, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 144, 148, and 154, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3.6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 156, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 157, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 372, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 377, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 373, and 152, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 378, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 374, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 593, 594, and 595, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 507, 513, and 517, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 604, 605, and 606, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 288, 290, and 292, respectively; or a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 289, 291, and 293, respectively.
[0018] [0018] In some modalities of any of the polynucleotides described here, the Vx region contains a CDR-H1, CDR-H2 and
[0019] [0019] In some embodiments of any of the polynucleotides described herein, the Vx region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247- 256, 324, 325, 518- 531, 533, 609, 617, 772-774, or 814-832. In some respects, the Vx region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617. In some embodiments, the Vx region contains a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 593, 594, and 595 , respectively; or the Vu region includes CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively. In some modalities, the Vy region is or includes the amino acid sequence mentioned in SEQ ID NO: 617.
[0020] [0020] In some modalities of any of the polynucleotides described here, region V. includes CDR-L1, CDR-L2 and CDR-L3 selected from: a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 28, 39, and 49, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 29, 40, and 50, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the
[0021] [0021] In some modalities of any of the polynucleotides described here, region V. includes CDR-L1, CDR-L2 and CDR-L3 selected from: a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 28, 39, and 49, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 30, 39, and 51, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 31, 41, and 52, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 33, 43, and 55, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 34, 44, and 56, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 185, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 189, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 176, 181, and 190, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 177, 182, and 191, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 192, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 178, 183, and 193, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 178, 183, and 194, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 30, 399, and 415, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 380, 400, and 416, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 33, 43, and 421, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 381, 401, and 417, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the
[0022] [0022] In some of these modalities, the V. region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610,618, 775 - 777, or 833-849. In some respects, the V. region is or contains the amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266 , 267, 326, 327, 534, 535, 536, 537, 538, 610,618, 775-777, or 833-849.
[0023] [0023] In some embodiments of any of the polynucleotides described herein, region V. contains a CDR-L1, CDR-L2 and CDR-L3 including the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; or the V. region contains a CDR-L1, CDR-L2 and CDR-L3 including the amino acid sequence of SEQ ID NOs: 614, 615, and 603, respectively. In some cases, the V. region is or includes the amino acid sequence mentioned in SEQ ID NO:
[0024] [0024] In some of the modalities, the Vx region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the Vi region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534- 550, 552-557, 610, 775-777, or 833-849.
[0025] [0025] In some of the modalities, the Vu region is either comprised of CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vu region selected from any of SEQ ID NOs: 617 , 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and region V. is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 618, 116-127, 257- 267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
[0026] [0026] In some of the modalities, the Vx region is or comprises (a) a CDR-H1 that comprises the selected sequence from any one of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289, 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 that comprises the selected sequence from any one of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 that comprises the sequence selected from any one of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517 , or 606; and region V. is or comprises (a) a CDR-L1 that comprises the sequence selected from any one of SEQ ID NOs: 601, 614, 26-36, 174- 178, 302, 303, 380- 392, 394-398, 589, or 607; (b) a CDR-L2 that comprises the selected sequence from any one of SEQ ID NOs: 602, 615, 37-46, 179-183, 304, 305, 399-409, 411-414, 590, or 608 ; and (c) a CDR-L3 that comprises the selected sequence
[0027] [0027] In some of these modalities, the Vx region and the VL regions include the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 116, respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively, or an amino acid sequence that has at least 90% identity for SEQ ID NO: 111 and 117 , respectively; the Vu region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 118, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 119, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 119, respectively ; the Vr region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 120, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 120, respectively ; the Vn region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 121, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 121, respectively ; the Ve region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 122, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 122, respectively; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 123, respectively, or an amino acid sequence that has at least
[0028] [0028] In some embodiments of any of the polynucleotides described here, the Vu region and the V regions encoded by the polynucleotides include the amino acid sequence mentioned in
[0029] [0029] In some of the modalities, the Va region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247- 256, 324, 325, 518-531, 533, 609, 772-774 , or 814-832; and the V. region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-
[0030] [0030] In some embodiments of any of the polynucleotides described here, the fragment includes a scFv. In some modalities, the Vu region and the V. region are joined by a flexible ligand. In some embodiments, scFv includes a linker containing the amino acid sequence GGGGSGGGGSGCGGGGS (SEQ ID NO: 361). In some embodiments, the Vn region is amino-terminal to the V region.
[0031] [0031] In some embodiments of any of the polynucleotides described herein, the antigen-binding domain includes the amino acid sequence selected from any of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity for the selected amino acid sequence from any of SEQ ID NOs: 128-139, 268-278, 329,
[0032] [0032] In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the antigen binding domain includes (a) the nucleotide sequence mentioned in any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOS: 330-352,647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen binding domain includes (a) the nucleotide sequence mentioned in any of SEQ ID NOS: 352, 647, 648, 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOS: 352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen-binding domain is codon-optimized. In some embodiments, the nucleic acid encoding the antigen-binding domain includes the nucleotide sequence established in any of SEQ ID NO: 440, 460, 715, 717 or 719. In some embodiments, the nucleic acid that encodes the antigen-binding domain includes the nucleotide sequence mentioned in SEQ ID NO: 460.
[0033] [0033] In some modalities of any of the polynucleotides,
[0034] [0034] Chimeric antigen receptors are provided, comprising: (1) an extracellular antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA), in which the antigen-binding domain extracellular structure comprises: (i) a variable heavy chain (Vs) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % sequence identity for the sequence of the Vx region of SEQ ID NO: 617; and (ii) a variable region of the light chain (V.) comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98 % or 99% sequence identity for the V region sequence of any of SEQ ID NO: 618; (2) a spacer mentioned in SEQ ID NO: 649 or where the nucleic acid encoding the spacer is or comprises the sequence mentioned in SEQ ID NO: 622; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta chain (CD37) and an intracellular signaling domain of a T cell co-stimulating molecule. polynucleotides that encode that chimeric antigen receptor are provided. In some embodiments, the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 contained in the sequence of the Vn region of SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained in the sequence of the V. region of SEQ ID NO: 618; or the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 593, 594 and 595, respectively, and the Vi region. comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 596, 597 and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2 and CDR - L3 comprising the sequence of SEQ ID NOS: 601, 602 and 603, respectively; the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 598, 599 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 601, 602 and 603, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 611, 612 and 613, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the sequence of SEQ ID NOS: 614, 615 and 603, respectively.
[0035] [0035] Chimeric antigen receptors are provided, comprising: (1) an extracellular antigen binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen binding domain comprises: a variable heavy region (Vx4) comprising a CDR-H1, CDR-H2 and CDR-H3 contained within the Vx region sequence of SEQ ID NO: 617; and the light variable region (V.) comprising CDR-L1, CDR-L2 and CDR-L3 contained within the V. region sequence of SEQ ID NO: 618; or the Va region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the V4 region sequence of SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. region sequence of SEQ ID NO: 618; or the Vx region comprised of CDR-H1, CDR-H2 and CDR-H3 that comprise the sequence of
[0036] [0036] In some embodiments, the receptor includes an antigen-binding domain that binds to the same or substantially the same epitope in BCMA, or competes for binding to BCMA with any of the antibodies and fragments or antibodies with the combinations provided of VH / VL or CDR sequences, described herein, including in any of the previous modalities. In some embodiments, the binding domain recognizes an epitope comprising a portion of one or more amino acid sequences within a BCMA polypeptide. In some respects, one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO: 640), YFDSL (SEQ ID NO: 779) and QLRCSSNTPPL (SEQ ID NO: 642). In some respects, one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO: 640), YFDSLL (SEQ ID NO: 641) and QLRCSSNTPPL (SEQ ID NO: 642). In some respects, one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO: 640), QNEYFDSLL (SEQ ID NO: 780) and QLRCSSNTPPL (SEQ ID NO: 642). In some respects, one or more amino acid sequences are or comprise: QNEYF (SEQ ID NO: 637), CI-PCQL (SEQ ID NO: 638) and CORYC (SEQ ID NO: 639). In some aspects, one or more amino acid sequences are or comprise: CSQNEYF (mentioned in SEQ ID NO: 410) and LLHACIPCQLR (mentioned in SEQ ID NO: 428).
[0037] [0037] In some modalities of any of the polynucleotides described here, the intracellular signaling region includes an activation cytoplasmic signaling domain. In some modalities, the activation cytoplasmic signaling domain is able to induce a primary activation signal in a T cell, is a T cell receptor (TCR) and / or includes a tyrosine-based activation motif and immunoreceptor (ITAM). In some embodiments, the activation cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta chain (CD376) or a functional variant or signaling portion thereof. In some embodiments, the cytoplasmic activation domain is human or is derived from a human protein. In some embodiments, the cytoplasmic activation domain is or includes a sequence mentioned in SEQ ID NO: 628 or an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 628.
[0038] [0038] In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the cytoplasmic activation domain is or includes a sequence mentioned in SEQ ID NO: 627 or is a codon-optimized sequence and / or de- generated sequence. In other embodiments, the nucleic acid encoding the activation cytoplasmic signaling domain is or includes a sequence mentioned in SEQ ID NO: 652.
[0039] [0039] In some embodiments of any of the polynucleotides described here, the intracellular signaling region also includes a co-stimulating signaling region. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a portion of the signaling thereof. In some embodiments, the co-stimulating signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the co-stimulating signal region is human or is derived from a human protein. In other embodiments, the co-stimulatory signal region is or includes a sequence mentioned in SEQ ID NO: 626 or an amino acid sequence that exhibits at least 90% sequence identity for the sequence mentioned in SEQ ID NO: 626.
[0040] [0040] In some embodiments of any of the polynucleotides described here, the nucleic acid encoding the co-stimulated region
[0041] [0041] In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the transmembrane domain is or includes a sequence mentioned in SEQ ID NO: 623 or is a codon and / or sequence optimized sequence degenerated. In some embodiments, the nucleic acid encoding the transmembrane domain includes the sequence mentioned in SEQ ID NO:
[0042] [0042] In some of the modalities, the polynucleotide comprises the sequence mentioned in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86%, 87%,
[0043] [0043] In some embodiments, the polynucleotide further encodes a truncated receptor.
[0044] [0044] Vectors are also provided comprising any of the polynucleotides described herein. In some embodiments, the vector is a viral vector. In some modalities, the viral vector is a retroviral vector. In some modalities, the viral vector is a slow vector.
[0045] [0045] In some respects, chimeric antigen antigens encoded for a polynucleotide of any of the modalities described here are provided. In some embodiments, the chimeric antigen receptor includes: (a) an extracellular antigen binding domain that specifically recognizes the B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.
[0046] [0046] In some embodiments of any of the chimeric antigen receptors described here, the spacer is derived from an immunoglobulin. In some embodiments, the spacer includes a sequence of a joint region, a region of Ch2 and Cn3. In some modalities of any of the chimeric antigen receptors described here, one of the joint, CHx2 and CH3, is derived wholly or partially from IgG4 or IgG2. In some modalities, the joint, Ch2 and Cx3 is derived from IgG4. In some embodiments, one or more of the Cx2 and Ch3 joints are chimeric and include the sequence derived from IgG4 and IgG2. In some modes, the spacer includes an Ig9G4 / 2 chimeric joint, an IgG2 / 4 CH2 region and an IgG4 CH3 region.
[0047] [0047] In some embodiments of any of the chimeric antigen receptors described herein, the spacer is or includes (1) the sequence mentioned in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 649; or (iii) a contiguous portion of (i) or (ii) with at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes a sequence mentioned in SEQ ID NO: 649.
[0048] [0048] Other chimeric antigen receptors are provided that include (a) an extracellular antigen binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) a signaling region
[0049] [0049] In some embodiments of any of the chimeric antigen receptors described herein, the Vu region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256,324, 325, 518-531, 533, 609, 617, 772- 774, or 814-832; and / or the V region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849 .
[0050] [0050] In some embodiments of any of the chimeric antigen receptors described herein, the Vu region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and / or the VL region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534 , 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
[0051] [0051] In some modalities of any of the chimeric antigen receptors described here, the Vu region is or includes the
[0052] [0052] In some modalities of any of the chimeric antigen receptors described here, the Vn region is or includes a CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vx region selected a from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772 -774 or 814-832; and / or the V. region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 116, 117, 118 , 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610,618, 77T5-777, or 833-849.
[0053] [0053] In some embodiments of any of the chimeric antigen receptors described herein, the Vx region is or includes (a) a heavy chain 1 complementarity determining region (CDR-H1) containing the selected amino acid sequence a from any one of SEQ ID NOs: 1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) containing the selected amino acid sequence from any of SEQ ID NOs: 4-6, 145-148, 290, 291 , 296, 297,372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain 3 complementarity determining region (CDR-H3) containing the selected amino acid sequence from any of SEQ ID NOs: 7-
[0054] [0054] In some embodiments of any of the chimeric antigen receptors described herein, the Vx region is or includes (a) a heavy chain 1 complementarity determining region (CDR-H1) containing the selected amino acid sequence a from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604,611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) containing the selected amino acid sequence from any of SEQ ID NOs: 4, 5, 6, 145, 147, 148 , 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain 3 complementarity determining region (CDR-H3) containing the selected amino acid sequence from any of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155 , 156, 157, 292, 293, 376, 517,595, 606 or 613; and / or the V. region is or includes (a) a light chain 1 complementarity determining region (CDR-L1) containing the amino acid sequence selected from any of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determination region 2 (CDR-L2) containing the selected amino acid sequence from any of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602,608 or 615; and (c) a light chain 3 complementarity determining region (CDR-L3) containing the amino acid sequence selected from any of SEQ ID NOs: 47, 48, 49, 51, 52, 55 , 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418,421, 591, or 603. In some embodiments of any of the chimeric antigen receptors described herein, the V region " includes CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 1.4, and 7, respectively, a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the sequence - amino acid class of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5 and 10, respectively; a CDR -H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3,6, and 11, respectively; a CDR-H1, CDR- H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 140, 145 and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 141, 145, and 150, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 142, 146, and 151, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 152, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 143, 147, and 153, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 144, 148, and 154, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 156, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 157, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 372, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 377, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 373, and 152, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5 and 378, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 374, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 593, 594, and 595, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 507, 513, and 517, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 604, 605, and 606, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 288, 290, and 292, respectively; or a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence
[0055] [0055] In some modalities of any of the chimeric antigen receptors described here, the Vn region includes CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 1.4, and 7, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 10, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 143, 147, and 153, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 144, 148, and 154, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 156, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 5, and 157, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 2, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 155, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 372, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 3, 6, and 376, respectively; a CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 593, 594, and 595, respectively; a CDR-H1,
[0056] [0056] In some embodiments of any of the chimeric antigen receptors described herein, the Vu region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518 -531, 533, 609,617, 772-774, or 814-832. In some embodiments of any of the chimeric antigen receptors described herein, the Vn region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609,617, 772-774, or 814-832. In some embodiments, region V "includes CDR-H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 593, 594, and 595, respectively; or the Vr region includes CDR- H1, CDR-H2 and CDR-H3 containing the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively In some embodiments, the Vn region is or includes the amino acid sequence mentioned in SEQ ID NO: 617.
[0057] [0057] In some modalities of any of the chimeric antigen receptors described here, region V. includes CDR-L1, CDR-L2 and CDR-L3 selected from: a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively
[0058] [0058] In some modalities of any of the chimeric antigen receptors described here, region V. includes CDR-L1, CDR-L2 and CDR-L3 selected from: a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 28, 39, and 49, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 30, 39, and 51, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 31.41, and 52, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 33, 43, and 55, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 34, 44, and 56, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 185, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 189, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 176, 181, and 190, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 177, 182, and 191, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 174, 179, and 192, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 178, 183, and 193, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 178, 183, and 194, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 30, 399, and 415, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 380, 400, and 416, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 33, 43, and 421, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 381, 401, and 417, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 382, 402, and 418, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 614, 615, and 603, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 589, 590, and 591, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 607, 608, and 591, respectively; a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2 and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 303, 305, and 307, respectively.
[0059] [0059] In some embodiments of any of the chimeric antigen receptors described herein, the V. region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610,618, 775-777, or 833-849. In some embodiments of any of the chimeric antigen receptors described herein, the V. region is or includes the amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262 , 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610,618, 775-777, or 833-849.
[0060] [0060] In some modalities of any of the chimeric antigen recipients described here, region V. includes CDR-
[0061] [0061] In some modalities of any of the chimeric antigen receptors described herein, the V. region is or includes the amino acid sequence mentioned in SEQ ID NO: 618. In some modalities of any of the antigen receptors chimerics described herein, the Vx region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO : 110 and 116, respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 111 and 117, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 118, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 119, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 119, respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 120, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 120, respectively ; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 121, respectively, or an amino acid sequence that has at least 90% identity for SEQ ID NO: 110 and 121 , respectively; the Vu region and
[0062] [0062] In some modalities of any of the chimeric antigen receptors described here, the Vx region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence that has at least 90% identity for SEQ ID NO: 110 and 116, respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively, or an amino acid sequence that has at least 90% identity for SEQ ID NO: 111 and 117 , respectively; the Vu region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 118, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 120, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 120, respectively ; the Vr region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 110 and 121, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 121, respectively ; the Vy region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 112 and 124, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 112 and 124, respectively ; the Ve region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 113 and 125, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 113 and 125, respectively; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 248 and 258, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 248 and 258 , respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 252 and 262, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 252 and 262, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 253 and 263, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 253 and 263, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 254 and 264, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 254 and 264, respectively; the Vu region and the VL regions contain the amino acid sequence mentioned in SEQ ID NOs: 255 and 265, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 255 and 265, respectively; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 256 and 266, respectively, or an amino acid sequence that has at least 90% identity for SEQ ID NO: 256 and 266 , respectively; the Vu region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 256 and 267, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 256 and 267, respectively ; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 518 and 534, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 518 and 534, respectively ; the Vr region and the V regions contain the amino acid sequence mentioned in SEQ ID NOs: 519 and 535, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 519 and 535, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 115 and 536, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 115 and 536, respectively ; the Ve region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 520 and 264, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 520 and 264, respectively; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 521 and 537, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 521 and 537 , respectively; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 522 and 538, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 522 and 538, respectively ; the Vu region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 609 and 610, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 609 and 610, respectively ; the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 617 and 618, respectively; the Vn region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 324 and 326, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 324 and 326, respectively ; or the Vx region and the V. regions contain the amino acid sequence mentioned in SEQ ID NOs: 325 and 327, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 325 and 327, respectively.
[0063] [0063] In some embodiments of any of the chimeric antigen receptors described here, the fragment includes an scFv. In some embodiments, the Vu region and the V. region are joined by a flexible ligand. In some embodiments, scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 361). In some embodiments, the Vx region is amino-terminal to the V region.
[0064] [0064] In some embodiments of any of the chimeric antigen receptors described herein, the antigen-binding domain includes the amino acid sequence selected from any of SEQ ID NOs: 128-139, 268-278, 329 , 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% sequence identity for the selected amino acid sequence from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585 , or 769-771. And bad-
[0065] [0065] In some modalities of any of the chimeric antigen receptors described here, the Vx region is carboxy-terminal to the V1 region. In some embodiments, scFv includes the amino acid sequence mentioned in SEQ ID NOs: 328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity for the amino acid sequence mentioned in SEQ ID NO: 328 or
[0066] [0066] In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region includes an activation cytoplasmic signaling domain. In some embodiments of any of the chimeric antigen receptors described here, the activation cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a component of the T cell receptor (TCR) and / or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the activation cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta chain (CD3C0) or a functional variant or signaling portion thereof. In some modalities, the cytoplasmic activation domain is human or is derived from a human protein. In some modalities, the cytoplasmic domain
[0067] [0067] In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region further includes a co-stimulating signaling region. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the co-stimulatory signaling region is human or is derived from a human protein. In some embodiments, the co-stimulatory signal region is or includes a sequence mentioned in SEQ ID NO: 626 or an amino acid sequence that exhibits at least 90% sequence identity for the sequence mentioned in SEQ ID NO: 626. In in some modalities, the co-stimulating signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28 or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some modes, the transmembrane domain is human or is derived from a human protein. In some embodiments of any of the chimeric antigen receptors described herein, the transmembrane domain is or includes a sequence mentioned in SEQ ID NO: 624 or an amino acid sequence that exhibits at least
[0068] [0068] In some embodiments of any of the chimeric antigen receptors described here, the encoded chimeric antigen receptor includes its N to C-terminal in order: the antigen-binding domain, the spacer, the transmembrane domain , and the intracellular signaling domain.
[0069] [0069] In some of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence that comprises the sequence mentioned in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the sequence mentioned in any of SEQ ID NOS: 751-756. In some of any of the modalities, the chimeric antigen receptor is encoded by a polynucleotide sequence that comprises the sequence mentioned in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the sequence mentioned in any of SEQ ID NOS: 755 and 756. In some of any of the modalities, the chimeric antigen receptor is encoded by a polynucleotide sequence that comprises the sequence mentioned in SEQ ID NO: 755 or a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity following this. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence that comprises the sequence mentioned in SEQ ID NO: 755.
[0070] [0070] Modified cells containing a polynucleotide of any of the modalities described here are provided in some modalities. In some embodiments of any of the modified cells described herein, the modified cell contains the chimeric antigen receptor of any of the embodiments described herein.
[0071] [0071] In some embodiments of any of the modified cells described here, the cell is an immune cell. In some embodiments, the immune cell is a primary cell obtained from an individual. In some embodiments, the immune cell is an NK cell or a T cell. In some embodiments, the immune cell is a T cell, and the T cell is a CD4 + and / or CD8 + T cell.
[0072] [0072] In some embodiments of any of the modified cells described here, the cell contains transcribed RNA that encodes the chimeric antigen receptor, optionally messenger RNA (MRNA), which exhibits at least 70%, 75%, 80%, 85%, 90% or 95% homogeneity of RNA. In some embodiments, the cell contains transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mMRNA), which exhibits reduced heterogeneity compared to the heterogeneity of the mMRNA transcribed in a cell encoding a chimeric antigen receptor of reference, said reference chimeric antigen receptor containing the same amino acid sequence as the chimeric antigen receptor, but encoded by a different polynucleotide sequence containing one or more nucleotide differences in the polynucleotide encoding the CARs and / or in which the reference chimeric antigen receptor is encoded by a polynucleotide that contains one or more binding donor sites and / or one or more binding receptor sites in the nucleic acid encoding the spacer. In some modalities, the RNA heterogeneity is reduced by greater or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some embodiments, the cell encoding the reference CAR includes the transcribed RNA encoding the reference CAR, optionally the messenger RNA (mMRNA), which exhibits more or more than about 10%, 15%, 20%, 25 %, 30%, 40%, 50% or more of RNA heterogeneity. In some modalities, RNA homogeneity and / or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation or liquid chromatography.
[0073] [0073] In some modalities of any of the modified cells described here, among a plurality of modified cells, less than or less than about 10%, 9%, 8%, 7%, 5%, 4%, 3% , 2% or 1% of cells in the plurality contain a chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.
[0074] [0074] Compositions are also provided which comprise any of the modified cells provided herein. In some of these embodiments, the composition comprises CD4 + and CDB8 + T cells, and the ratio of CD4 + T cells to CD8 + is about 1: 3 to 3: 1.
[0075] [0075] Compositions containing a polynucleotide of any of the modalities described herein, a chimeric antigen receptor of any of the modalities described herein, or a modified cell of any of the modalities described herein are also provided here. In some embodiments, the composition further contains a pharmaceutically acceptable excipient. In some of these modalities, the composition is sterile.
[0076] [0076] In other respects, treatment methods are provided that involve the administration of modified cells of any of the modalities described herein or the composition of any of the modalities described herein to an individual with a disease or disorder. In some embodiments, the method comprises administering a dose of the modified cells or a composition comprising a dose of the modified cells.
[0077] [0077] Uses of any of the modified cells or the compositions described herein are also provided for the manufacture of a medicament for the treatment of a disease or disorder. Uses of any of the modified cells or the compositions described herein are also provided for the treatment of a disease or disorder. In some of these embodiments, the modified cells or composition are for use in a treatment regimen, where the treatment regimen comprises administering a dose of the modified cells or a composition comprising a dose of the modified cells.
[0078] [0078] In some modalities of any of the methods described here, the disease or disorder is associated with the expression of B cell maturation antigen (BCMA). In some modalities, the disease or disorder associated with BCMA is a disorder related to the B cell. In some modalities, the disease or disorder associated with BCMA is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA-associated vasculitis, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic thrombosis (TTP), TTP), autoimmune thrombocytopenia, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarthritis, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, IgM polyneuropathies , diabetes mellitus, Reynaud's syndrome, antiphospholipid syndrome, Goodpasture's disease, Kawasaki's disease, autoimmune hemolytic anemia, myasthenia gravis or progressive glomerulonephritis.
[0079] [0079] In some modalities of any of the methods described here, the disease or disorder associated with BCMA is a cancer
[0080] [0080] In some of the modalities, the dose of modified T cells comprises between or about 1 x 107 T cells of CAR expression and the or about 2 x 10 T cells of CAR expression. In some of the modalities, the dose of modified T cells comprises between or about 2.5 x 10 7 T cells of CAR expression and at or about 1.2 x 10 T cells of CAR expression, between or about 5.0 x 107 T cells of CAR expression and at or about 4.5 x 10º T cells of CAR expression, or between or about 1.5 x 10 T cells of CAR expression and a or about 3.0 x 10º T cells of CAR expression. In some embodiments, the dose of modified T cells comprises at or about 2.5 x 10 7, at or about 5.0 x 10 7, at or about 1.5 x 10 8, at or about 3.0 x 108, at or about 4.5 x 108, at or about 8.0 x 10º or at or about 1.2 x 10º T cells of CAR expression. In some embodiments, the dose of modified T cells comprises at or about 5.0 x 10, at or about 1.5 x 108, at or about 3.0 x 10 ° or at or about 4.5 x 10 ° CAR expression T cells. In some of the modalities, the dose of modified T cells comprises a combination of CD4 * T cells and CD8 * T cells, in a defined ratio of CAR expression CD4 * T cells to CAR expression CD8 * T cells and / or from CD4 * T cells to CD8 * T cells, which is either approximately 1: 1 or approximately 1: 3 to approximately 3: 1.
[0081] [0081] In some of the modalities, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of CAR expression T cells in a dose of modified T cells express an apoptosis marker, optionally Annexin V or active Caspase 3. In some of the modalities, less than 5%, 4%, 3%, 2% or 1% of CAR expression T cells in a dose of modified T cells express Annex V or active Caspase 3.
[0082] [0082] In some of the modalities, before the administration, the individual received a lympho-exhaustion therapy, which includes the administration of fludarabine at or about 20-40 mg / m body surface area of the individual, optionally at or about 30 mg / m , daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m body surface area of the individual, optionally at or about 300 mg / m , daily, for 2-4 days.
[0083] [0083] In some of the modalities, the individual received a lymphodepletation therapy that comprises the administration of fluudabine at or about 30 mg / m of the individual's body surface, daily and cyclophosphamide at or about 300 mg / m2 of the body surface
[0084] [0084] In some modalities, on or before the administration of the cell dose, the individual received three or more therapies selected from: autologous stem cell transplantation (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody.
[0085] [0085] In some modalities, the immunomodulating agent is selected from thalidomide, lenalidomide or pomalidomide. In some modalities, the proteasome inhibitor is selected from among bortezomib, carfilzomib or ixazomib. In some embodiments, the anti-CD38 antibody is or comprises daratumumab.
[0086] [0086] In some modalities, in administering the cell dose, the individual had no active history or plasma cell leukemia (PCL).
[0087] [0087] In some modalities, when administered to individuals, the dose or composition is able to achieve an objective response (OR), in at least 50%, 60%, 70%, 80%, 80%, 90% or 95% of individuals who were administered. In some modalities, OR includes individuals who achieve a strict complete response (sSCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimum response (MR). In some modalities, when administered to individuals, the dose or composition is capable of obtaining a strict complete response (SCR), complete response (CR), very good partial response (VGPR) or partial response (PR), at least 50 %, 60%, 70%, 80% or 85% of the individuals who were administered. In some modalities, when administered to individuals, the dose or composition is capable of obtaining a strict complete response (SCR) or complete response (CR) of at least 20%, 30%, 40% 50%, 60% or 70% of subjects that were administered.
[0088] [0088] In some embodiments, the dose of modified T cells comprises about 5.0 x 107, about 1.5 x 108, about 3.0 x 10º or about 4.5 x 10º T cells of expression of CAR. In some embodiments, the dose of the modified T cells comprises about 5.0 x 10 7 CAR expression T cells.
[0089] [0089] Methods are also provided herein to determine the heterogeneity of a nucleic acid transcribed from a transgene, the method comprising: a) amplifying a transcribed nucleic acid using at least one pair of 5 'and 3' primers, wherein at least one pair comprises a 5 'primer that is complementary to a nucleic acid sequence in the 5' untranslated region (5 'UTR) of the transcribed nucleic acid and a 3' primer that is complementary to an acid sequence nucleic in the 3 'untranslated region (3' UTR) of the transcribed nucleic acid to generate one or more amplified products; and b) detecting the amplified products, in which the presence of two or more amplified products from at least one pair of 5 'and 3' primers indicates heterogeneity in the amplified products.
[0090] [0090] In some modalities of the method, the differences detected in b) are different lengths of the amplified transcripts. In some embodiments, the differences in b) are differences in the chromatographic profiles of the amplified transcripts.
[0091] [0091] In some modalities, differences in amplified products are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation or chromatography. In some modalities, the 5 'primer is specific for the transcribed sequence of the transcribed nucleic acid promoter region. In some embodiments, the transcribed nucleic acid is amplified using a 3 'primer specific for a sequence within the polynucleotide amino acid coding sequence and / or the 3' untranslated region,
[0092] [0092] In some embodiments, step a) is performed by a single amplification reaction, using a single 5 'and 3' primer pair comprising a 5 'primer that is complementary to a nucleic acid sequence within the region untranslated into 5 '(5' UTR) of the transcribed nucleic acid and a 3 'primer that is complementary to a nucleic acid sequence within the untranslated 3' (3 'UTR) region. In some embodiments, step a) is carried out by parallel or subsequent amplification reactions using a first 5 'and 3' primer pair, a second 5 'and 3' primer pair and, optionally, additional 5 'and 3 ', wherein: the first polymer 5' and 3 'pair contains a 5' primer that is complementary to a nucleic acid sequence within the 5 'RTU of the transcribed nucleic acid and a 3' primer that is complementary to a nucleic acid sequence within the 3 'UTR of the transcribed nucleic acid; the second pair of 5 'and 3' primers contains a 5 'primer whose sequence is complementary to a portion of the transduced sequence of the nucleic acid transcription and a 3' primer whose sequence is complementary to an acid sequence nucleic within the 3 'UTR of the transcription; and the optional additional 5 'and 3' pairs contain sequences complementary to the sequences within the translated region of the transcription. In some modalities, parallel or subsequent amplification reactions amplify overlapping parts of the transcript.
[0093] [0093] In some modalities, amplified products are expected to have about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 kilobases, 4.5 kilobases, 5 kiloba - ses, 5.5 kilobases, 6 kilobases, 7 kilobases or 8 kilobases in length.
[0094] [0094] In some embodiments, a transcribed nucleic acid that is detected as having heterogeneity is identified as a candidate for transgene to remove one or more binding sites. In some embodiments, the transgene candidate's transcribed nucleic acid exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity after expression in a cell.
[0095] [0095] Methods are also provided to reduce the heterogeneity of an express transgene transcription, the method comprising: a) identifying a transgene candidate for the removal of binding sites according to any of the methods for determining terminating the heterogeneity of a transcribed nucleic acid provided herein; b) identify one or more potential binding donors and / or binding receptor sites; and c) modifying the nucleic acid sequence at or near one or more potential binding donor sites and / or binding receptors identified in b), thereby generating a modified polylucleotide.
[0096] [0096] In some of these modalities, the method also involves d) evaluating the application of transgene for the removal of binding sites, as in step a). In some of these modalities, the method also involves e) repeating steps b) -d) until the heterogeneity of transcription in step d) is reduced compared to the heterogeneity of transcription, as determined in step a) .
[0097] [0097] In some of these modalities, the one or more potential binding donor sites and / or binding receptors display a score of about or at least about 0.7, 0.75, 0.8, 0, 85, 0.9, 0.95 or 1.0 of a link event and / or is expected to be involved in a link event with a probability of at least 70%, 75%, 80% , 85%, 90%, 95%, 99% or 100%.
[0098] [0098] In some of these modalities, the donor binding sites and the receptor binding sites are identified independently. In some of these modalities, the recipient site (s) and / or binding donor (s) is / are canonical recipient site (s) and / or canonical donor (s) (s) and / or cryptic (s).
[0099] [0099] In some of these modalities, the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor. In some of these embodiments, the CAR polypeptide comprises an antigen binding domain comprising an antibody fragment, optionally a single chain antibody (scFv) fragment, comprising a variable heavy chain (Vr) and a variable light chain (V. ), a spacer (for example, a spacer region located between the ligand binding domain and the transmembrane domain of the recombinant receptor), a transmembrane region and an intracellular signaling region.
[00100] [00100] In some of these modalities, the modified polynucleotide is not modified within the coding sequence for the antigen-binding domain of the encoded CAR polypeptide. In some of these modalities, the encoded amino acid sequence of the transgene remains unchanged after modification of the polynucleotide. In some of these modalities, the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80%, 85%, 90% or 95% homogeneity after the expression of the unmodified polynucleotide in a cell.
[00101] [00101] In some of these modalities, the cell is a human cell. In some of these modalities, the cell is a T cell.
[00102] [00102] In some of these modalities, the method is a method implemented by computer and in which one or more steps a) -c) occur in an electronic device that comprises one or more processors and memory.
[00103] [00103] Computer systems comprising a processor and memory are also provided, the memory comprising instructions operable to make the processor perform one or more steps of the methods to reduce the heterogeneity of an expressed transgene transcription. Brief Description of Drawings
[00104] [00104] FIG.1Ae1B represent the results of an assay that assesses the heterogeneity of RNA as assessed by electrophoresis on an agarose gel. FIG. 1A represents the RNA heterogeneity of several anti-BCMA CARs, containing either a long spacer region (LS) or a shorter CD28 spacer region. FIG. 1B represents the RNA heterogeneity of three different anti-BCMA CAR coding sequences, containing the long spacer region (LS), before and after the optimization of the coding sequence and elimination of the binding site (O / SSE ).
[00105] [00105] AFIG.2 represents the results of an assay that evaluates the levels of CAR expression of BCMA-LS on the surface of transduced T cells before (not SSE) and after optimization (O / SSE), and elimination the binding site of the coding sequence.
[00106] [00106] FIG.3 represents the comparison of the transduction efficiency of lentiviral vectors that encode BMCA-LS CAR constructs and lentiviral vectors that encode BCMA-LS CAR constructions that have been codon-optimized and modified to eliminate define the expected connection sites (O / SSE).
[00107] [00107] AFIG.A4A shows the results of an assay that assesses the cytolytic activity of BMCA-LS CAR expression T cells against cell lines expressing high (K562 / BCMA) or low (RPMI 8226) levels of BCMA in various effector relationships: target cells (E: T). FIG. 4B represents the cytolytic activity of several BMCA-LS CAR expression T cells against RPMI-8226 cells at an E: T ratio of 3: 1. FIG. 4C and FIG. 4D represent the cytolytic activity of non-optimized BCMA-LS CAR expression T cells and optimized BCMA-LS CAR expression T cells (O / SSE) in various BCMA expression cell lines.
[00108] [00108] FIG.5A represents the results of an assay that assesses the release of cytokines IFNy, I1L-2 and TNFa from BMCA-LS CAR expression T cells in response to incubation with cell lines that express high (K562 / BCMA) or low (RPMI 8226) levels of BOCMA in various effector: target cell (E: T) ratios (5: 1, 2.5: 1, 1.25: 1 and 0.6: 1 indicated as a, b, c and c, respectively, in the figure). FIG. 5B represents the release of IFNy, IL-2 and TNFa cytokines from unimproved BMCA-LS CAR expression T cells and optimized BCMA-LS CAR expression T cells (O / SSE) in response to incubation with K562 / BCMA and RPMI 8226 BCMA expression cells in different E: T ratios (3: 1, 1.5: 1, 0.75: 1 and 0.375: 1 indicated as a, b, c and c, respectively, in the figure ).
[00109] [00109] AFIG.6 represents the results of an assay that assesses cytolytic activity after incubation of BCMA-55-LS O / SSE CAR expression cells from two donors with BCMA expression cells that express variable levels of BCMA.
[00110] [00110] AFIG.7 represents the results of an assay that assesses the release of IFNy after incubation of BCMA-55-LS O / SSE CAR expression cells from two donors with BCMA expression cells that express varying levels of BCMA.
[00111] [00111] FIG.8 represents results of an assay that assesses the cytolytic activity of CAR T cells expressing anti-BCMA expressing CARs containing different spacer regions, in OPM2 target cells.
[00112] [00112] FIG.9A and 9B represent results of an assay that assesses the cytolytic activity of anti-CAR expression T cells
[00113] [00113] FIG. 10A represents the results of an assay that assesses the cytolytic activity of T cells expressing optimized anti-BCMA CAR (O / SSE) in the presence of supernatant from the H929 multiple myeloma cell line. FIG. 10B represents the results of an assay to assess the cytolytic activity of T cells expressing optimized anti-BCMA CAR (O / SSE) in the presence of recombinant B cell activation factor (BAFF).
[00114] [00114] FIG. 11A and 11B show results of an assay that assesses the release of IFNy, IL-2 and TNFa cytokines after incubation of anti-BCMA CAR expression T cells with OPM2 target cells in the presence of soluble BCMA-Fc (FIG. 11A ) or supernatant from an H929 multiple myeloma cell line (FIG. 11B) in different concentrations (0 ng / ml, 111 ng / ml, 333 ng / ml and 1000 ng / ml indicated as a, b, ced , respectively, in the figures).
[00115] [00115] FIG. 12A represents the results of a trial that assesses tumor growth in a mouse model of human multiple myeloma xenograft of OPM 2, after a single intravenous injection of CAR T cells expressing optimized anti-BCMA CARs (O / SSE). FIG. 12B represents the results of a trial that evaluates survival in a mouse model of human multiple myeloma xenograft of OPM 2, after a single intravenous injection of CAR T cells expressing optimized anti-BCMA CARs (O / SSE).
[00116] [00116] AFIG.13A shows the results of an assay that evaluates tumor growth in an RPM / -8226 xenograft model (subcutaneous), after a single intravenous injection of CAR T cells expressing optimized anti-BCMA CAR (O / SSE). FIG. 13B represents survival in a mouse model of RPMI-8226 xenograft (subcutaneous), after a single intravenous injection of CAR T cells expressing optimized anti-BCMA CAR (O / SSE).
[00117] [00117] FIG. 14A and 14B represent results of an assay that assesses the number of CD4 + (FIG. 14A) and CD8 + (CD 14 +) CAR-positive T cells in the blood of RPMI-8226 xenograft mice (subcutaneous) treated with anti-CAR T cells -BCMA optimized (O / SSE) derived from a single donor (Donor 2).
[00118] [00118] FIG. 15A and 15B represent results of an assay that assesses the number of CD4 + (FIG. 15A) and CD8 + (CD 15 +) CAR-positive T cells in the blood of RPMI-8226 xenograft mice (subcutaneous) treated with anti-CAR T cells -BCMA optimized (O / SSE) derived from a single donor (Donor 1).
[00119] [00119] AFIG.16A shows the results of an assay that evaluates the expression level of tdTomato and a truncated receptor (substitute marker for CAR expression), detected by flow cytometry, in CAR expression cells of BCMA-55-LS-O / SSE, incubated for 6 hours in 96-well cell culture plates coated overnight with (0.008 pug / mL, 0.04 pg / mL, 0.2 pug / mL , 1 µg / ml and 5 µg / ml) of BCMA-Fc (soluble human BCMA fused at its C-terminus to an Fc region of the IgG fusion polypeptide). A recombinant Fc polypeptide was used as a control (CoFc Control).
[00120] [00120] FIG. 16B represents the results of an assay that assesses the percentage of tdTomato + cells among cells expressing the truncated receptor, in BCMA-55-LS-O / SSE CAR expression reporter cells, BCMA-26-LS-O CAR / SSE, BCMA-23-LS-O / SSE CAR and BCMA-25-LS-O / SSE CAR, incubated with (ten) 2-fold serial dilutions of BCMA-Fc. Cells that express a specific CAR for a different antigen (anti-CD19 CAR) were used as a control.
[00121] [00121] FIG.17 represents the percentage of tdTomato + cells among BCMA-55-LS-O / SSE CAR expression reporter cells or BCMA-55-SS CAR expression, after coculture with BCMA expression K562 target cells (BCMA.K562) in various E: T ratios.
[00122] [00122] FIG.18 represents the expression level of tdTomato and GFP (substitute marker for CAR expression), as detected by flow cytometry, in reporter cells expressing an anti-CD19 CAR, BCMA-55 CAR -LS-O / SSE, BCMA-26-LS- O / SSE CAR, BCMA-23-LS-O / SSE CAR or BCMA-52-LS- O / SSE CAR, incubated without antigen stimulation to evaluate the degree of antigen-independent (tonic) signaling for 3 days.
[00123] [00123] FIGS.19A and 19B represent the expression level of tdTomato and truncated receptor (substitute marker for CAR expression), as detected by flow cytometry, in reporter cells that express an anti-CD19 CAR, BCMA-55-LS-O / SSE, BCMA-26-LS-O / SSE CAR, BCMA-23-LS-O / SSE CAR or BCMA-52-LS-O / SSE CAR that contain intracellular domains derived from 4-1BB or CD28 incubated without antigenic stimulation to assess the degree of antigen-independent signaling (tonic).
[00124] [00124] FIG.20A represents the percentage of tdTomato + cells, evaluated by flow cytometry, among Nur77-tdTomato reporter cells modified to express BCMA-55-LS-O / SSE, specific for human BCMA, co-cultured with K562 human myeloid leukemia cells expressing human BCMA (huBCMA), murine BCMA (muBCMA) or cynomolgus monkey BCMA (cinoB-CMA), in the E: T ratio of 2: 1 or 5: 1. FIG. 20B and 20C represent the percentage (FIG. 20B), and the mean fluorescence intensity (MFI; FIG. 20C) of tdTomato + cells, assessed by flow cytometry, between BCMA-55-LS CAR expression reporter cells. -
[00125] [00125] FIG. 21A represents an exemplary amplification strategy for a transcript and a predicted amplified product. FIG. 21B represents exemplary amplified products resulting from the amplification of a transcription of known and unknown (cryptic) binding sites. FIG. 21C represents an exemplary sliding window amplification of a transcript using nested primer pairs. Detailed Description
[00126] [00126] Among the modalities provided are compositions, articles of manufacture, compounds, methods and uses, including those targeting or targeting cells and BCMA and BCMA expression diseases. It is observed that BCMA is expressed, for example, expressed heterogeneously, in certain diseases and conditions, such as malignancies or tissues or cells thereof, for example, in malignant plasma cells, as in all patients with recurrent or newly diagnosed eloma, for example, with little expression in normal tissues. Among the modalities provided, are methods useful in the treatment of such diseases and conditions and / or to target these cell types, including nucleic acid molecules that encode BCMA binding receptors, including antigen receptors. chimeric (CARs) and coded receivers, such as coded CARs, and compositions and articles of manufacture comprising them. Recipients can generally contain antibodies (including antigen-binding antibody fragments, such as variable heavy chain (Vr) regions, single domain antibody fragments, and single chain fragments, including scFvs) specific for BCMA . Cells are also provided, such as cells
[00127] [00127] Adoptive cell therapies (including those involving the administration of expression cells of specific chimeric receptors for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and / or other antigen receptors recombinants, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. In certain contexts, the methods available for adoptive cell therapy are not always fully satisfactory. In some respects, the ability of administered cells to recognize the attachment to a target, for example, target antigen, such as BCMA, to travel, locate and successfully enter appropriate sites within the individual, tumors and environments of the same so, to be activated, expand, exercise various effector functions, including cytotoxic death and secretion of various factors, such as cytokines, to persist, including in the long term, to differentiate, transition or engage in reprogramming in certain phenotypic states to provide effective and robust recall responses after clearance and re-exposure to the target ligand or antigen or to reduce exhaustion, anergy, terminal differentiation and / or differentiation in a suppressive state.
[00128] [00128] In some contexts, the ideal response to therapy may depend on the ability of modified recombinant receptors, such as CARs, to be expressed consistently and reliably on the cell surface and / or to bind the target antigen. For example, in some cases, the heterogeneity of the RNA transcribed from an introduced transgene (for example, encoding the recombinant receptor) can affect the expression and / or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy. In some contexts, the length, and the type of spacer in the recombinant receptor, such as a CAR, can affect the expression, activity and / or function of the receptor.
[00129] [00129] In addition, in some contexts, certain recombinant receptors may exhibit antigen-independent activity or signaling (also known as "tonic signaling"), which can lead to undesirable effects, such as increased differentiation and / or exhaustion of T cells expressing the recombinant receptor. In some ways, these activities may limit the activity, effect or potency of the T cell. In some cases, during modification, and the ex vivo expansion of cells for expression of the recombinant receptor, cells may exhibit indicative phenotypes. exhaustion, due to tonic signaling through the recombinant receptor.
[00130] [00130] In some contexts, properties of specific target antigens that recombinant receptors specifically bind, recognize or target, can affect receptor activity. In some contexts, B cell maturation antigen (BCMA) is typically expressed in malignant plasma cells and is an attractive therapeutic target for cell therapy. In some cases, BCMA can be cleaved by the gamma secretase, generating a soluble form of BCMA (sBCMA) or "spilled" from BCMA, reducing BCMA expressed on the surface of target cells. In some cases, the activity of BCMA-binding molecules, such as chimeric anti-BCMA antigen receptors, may be blocked or inhibited by the presence of soluble BCMA. Improved strategies are needed for optimal responses to cell therapies, in particular for recombinant receptors that specifically bind, recognize or target BCMA.
[00131] [00131] The modalities provided, in some contexts, are based on the observation that specific spacers and optimization of nucleic acid sequences can lead to consistent and robust expression of the recombinant receptor. The recombinant BCMA-binding receptors provided offer advantages over methods available for cell therapies, in particular, BCMA-directed cell therapy. In some modalities, as long as recombinant BCMA-binding receptors exhibit reduced tonic signaling independent of the antigen and lack of inhibition by soluble BCMA. In many respects, recombinant BCMA-binding receptors, polynucleotides encoding such receptors, modified cells and cellular compositions, exhibit certain desired properties that can overcome or neutralize certain limitations that can reduce ideal responses to cell therapy, for example. example, cell therapy with modified cells that express a recombinant BCMA-binding receptor. In some respects, it has been observed that compositions containing modified cells that express an exemplary BCMA-binding recombinant receptor shown here exhibit consistency in the cellular health of the modified cells and have been associated with the clinical response. In some contexts, the modalities provided, including recombinant receptors, polynucleotides that encode
[00132] [00132] In some respects, BCMA binding agents are provided, such as cell surface proteins, as recombinant receptors or receptors for chimeric antigens that bind to or recognize BCMA molecules and polynucleotides that encode surface proteins BCMA-binding cell, such as matching receptors (for example, CARs) and cells that express these receptors. BCMA-binding cell surface proteins generally contain antibodies (for example, antigen-binding antibody fragments) and / or other binding peptides that specifically recognize, how they specifically bind to BCMA, as BCMA proteins , like the human BCMA protein. In some aspects, the agent is the extracellular portion of BCMA.
[00133] [00133] In some modalities, polynucleotides are optimized or contain certain modified resources for optimization, such as using codons, to reduce RNA heterogeneity and / or modify, for example, increase or make it more consistent between batches of cellular products, expression, as a surface expression, of the coded receptor. In some embodiments, polynucleotides, which encode BCMA-binding cell surface proteins, are modified compared to a reference polynucleotide, such as to remove cryptic or hidden binding sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, which encode BCMA-binding cell surface proteins, are codon-optimized, as for expression in mammals, for example, a human cell, as in a human T cell. In some respects, modified polynucleotides result in an improved level of expression, for example, increased or more uniform or more consistent, for example, surface expression, when expressed in a cell. Such polynucleotides can be used in constructs for generating modified cells that express the encoded BCMA-binding cell surface protein. Thus, cells are also provided that express the recombinant receptors encoded by the polynucleotides provided here and use them in the therapy of adoptive cells, as a treatment for diseases and disorders associated with BCMA expression.
[00134] [00134] Among the polynucleotides provided are those that complicate recombinant receptors, such as antigen receptors, that specifically recognize, as they specifically bind, BCMA. In some respects, encoded receptors, such as those containing BCMA-binding polypeptides, in addition to compositions and articles of manufacture and uses thereof, are also provided.
[00135] [00135] Among the BCMA-binding polypeptides are antibodies, such as single chain antibodies (for example, antigen-binding antibody fragments) or portions thereof. In some examples, recombinant receptors are chimeric antigen receptors, such as those that contain anti-BCMA antibodies or their antigen-binding fragments. In either mode, an antibody or antigen-binding fragment, in the CARs provided, that specifically recognize an antigen, for example, BCMA, specifically binds to the antigen. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the recombinant encoded BCMA binding receptors.
[00136] [00136] In some cases, the polynucleotide encoding the BCMA-binding receptor contains a signal sequence that encodes a signal peptide; in some cases, encoded upstream of the nucleic acid sequences encoding the BCMA binding receptor or joined at the 5 'end of the nucleic acid sequences encoding the antigen binding domain. In some cases, the polynucleotide containing nucleic acid sequences that encode the BCMA-binding receptor, for example, chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some respects, the signal sequence can encode a signal peptide derived from a native polypeptide. In other respects, the signal sequence can encode a heterologous or non-native signal peptide. In some respects, the exemplary non-limiting signal peptide includes an IgG kappa chain signal peptide mentioned in SEQ ID NO: 620, or encoded by the nucleotide sequence mentioned in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain mentioned in SEQ ID NO: 851 and encoded by the nucleotide sequence mentioned in SEQ ID NO: 850; a CD8 alpha signal peptide mentioned in SEQ ID NO: 852; or a CD33 signal peptide mentioned in SEQ ID NO: 853. In some cases, the polynucleotide encoding the BCMA-binding receptor may contain nucleic acid sequence that encodes additional molecules, such as a substitute marker or other markers, or may contain additional components, such as promoters, regulatory elements and / or multicistronic elements. In some embodiments, the nucleic acid sequence encoding the BCMA-binding receptor can be operably linked to any of the additional components.
[00137] [00137] The BCMA-binding receptors provided generally contain an extracellular binding molecule and an intracellular signaling domain. Among the binding molecules provided are polypeptides containing antibodies, including single-chain cell surface proteins, for example, recombinant receptors, such as chimeric antigen receptors, containing those antibodies.
[00138] [00138] Among the binding molecules provided (for example, BCMA binding molecules) are single-stranded cell surface proteins, such as recombinant receptors (for example, antigen receptors), which include one of the antibodies provided or fragment thereof (eg BCMA binding fragment). Recombinant receptors include antigen receptors that specifically bind to or recognize BCMA, such as antigen receptors that contain the provided anti-BCMA antibodies, for example, antigen-binding fragments. Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Cells that express recombinant receptors and their use in adoptive cell therapy are also provided, as a treatment for diseases and disorders associated with BCMA expression.
[00139] [00139] Examples of antigen receptors, including CARs, and methods for modifying and introducing such receptors into cells, include those described, for example, in the publication of international patent application Nos. WO200014257, WOZ2013126726, WO 2012/129514, WO2014031687, WO2013166321, WO2013071154, WOZ2013123061 application publication Nos. US2002131960, US2013287748, US20130149337, United States Patent Nos./or those described by Sadelain et al /., Cancer Discov. April 2013; 3 (4): 388-398; Davila et a /. (2013) PLoS ONE 8 (4): e61338; Turtle et al, Curr. Opin. Immunol., 2012 October; 24 (5): 633-39; Wu et al., Cancer, 2012 March 18 (2): 160-75. In some respects, antenna receivers
[00140] [00140] In some embodiments, the CAR provided has an amino acid sequence selected from SEQ ID NOs: 757-762 or exhibits at least or about 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% sequence identity for the amino acid sequence mentioned in any of SEQ ID NOs 757-762. In some embodiments, the CAR provided is encoded by a polynucleotide, such as a polynucleotide with the nucleic acid sequence mentioned in any of SEQ ID NOs 751- 756, or a sequence that exhibits at least or at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the nucleic acid sequence mentioned in any of SEQ ID NOs: 751-756.
[00141] [00141] In some embodiments, the CAR provided is encoded by a polynucleotide, such as a polynucleotide with the nucleic acid sequence mentioned in any of SEQ ID NOs: 755 and 756, or a sequence that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the nucleic acid sequence mentioned in any of SEQ ID NOs: 755 and 756.
[00142] [00142] In some embodiments, the CAR provided is encoded by a polynucleotide, such as a polynucleotide with the nucleic acid sequence mentioned in SEQ ID NOs: 755 or a sequence that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the CAR provided is encoded by a polynucleotide, such as a polynucleotide with the nucleic acid sequence mentioned in SEQ ID NOs: 755.
[00143] [00143] In some embodiments, the nucleic acid encoding the antigen-binding domain comprises (a) the nucleotide sequence mentioned in any of SEQ ID NOS: 648, 330-352, 647, 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity with any of SEQ ID NOS: 648, 330-352, 647, 716 or 718; or (c) a degenerate sequence of (a) or (b).
[00144] [00144] Chimeric receptors include chimeric antigen receptors (CARs). Chimeric receptors, such as CARs, generally include an extracellular antigen-binding domain that includes, is or is comprised within, or comprises one of the provided anti-BCMA antibodies. Thus, chimeric receptors, for example, CARs, typically include in their extracellular portions one or more BCMA-binding molecules, such as one or more antigen-binding fragment, domain or portion, or one or more variable regions of the antibody and / or antibody molecules, as described herein.
[00145] [00145] The term "antibody" here is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and fragments of functional antibodies (antigen binding), including antigen binding fragments (Fab), F (ab ') fragments>, Fab' fragments, Fv fragments, recombinant IgG fragments (rlgG), heavy chain variable regions (Vn) capable of specifically binding to the antigen, fragments of single chain antibodies, included
[00146] [00146] The terms "complementarity determination region" and "CDR", synonymous with "hypervariable region" or "HVR", are known in the art for referring to non-contiguous amino acid sequences in the variable regions of antibodies, which confer specificity to the antigen and / or binding affinity. In general, there are three CDRs in each variable region of the heavy chain (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each variable region of the light chain (CDR-L1, CDR-L2, CDR-L3). "Structural regions" and "FR" are known in the art for referring to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each natural-sized heavy chain variable region (FR-H1, FR-H2, FR-H3 and FR-H4) and four FRs in each natural-sized light chain variable region (FR-L1 , FR-L2, FR-L3 and FR-L4).
[00147] [00147] The precise limits of the amino acid sequence of a given CDR or FR can be easily determined using any of several well-known schemes, including those described
[00148] [00148] The limits of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering for the Kabat and Chothia schemes is based on the most common antibody region string lengths, with entries accommodated by insertion letters, for example, "30a" and exclusions that appear in some antibodies. The two schemes place certain insertions and exclusions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on the analysis of complex crystal structures and is similar in many respects to Chothia's numbering scheme. The APM scheme is a compromise between the definitions of Kabat and Chothia, based on the one used by Oxford Molecular's AbM antibody modeling software.
[00149] [00149] Table 1, below, lists exemplary position limits for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, as identified by the schemes of Kabat, Chothia, ADM and Contact, respectively. For CDR-H1, the waste numbering is listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so on against. Note that, as the Kabat numbering scheme shown places inserts in H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention shown varies between H32 and H34, depending on the length of the shoulder strap.
[00150] [00150] Thus, unless otherwise specified, a "CDR" or "complementary determination region" or specified CDRs
[00151] [00151] Likewise, unless otherwise specified, a specified FR or FR (s) (for example, FR-H1, FR-H2, FR-H3, FR-H4) from a a given antibody or region, as a variable region, should be understood as encompassing a region of structure (or the specific one) as defined by any of the known schemes. In some cases, the scheme for identifying a specific CDR, FR or FR or CDRs is specified, such as the CDR, as defined by the method of Kabat, Chothia, ADM or Contact, or other known schemes. In other cases, the specific amino acid sequence of a CDR or FR is given.
[00152] [00152] The term "variable region" or "variable domain" refers to the domain of a heavy or light chain antibody that is involved in binding the antibody to the antigen. The heavy chain and light chain variable regions (Vn and V., respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single Vx or V. domain may be sufficient to confer antigen binding specificity. antibodies that bind a specific antigen can be isolated using a Vu or V domain from an antibody that binds the antigen to screen a library of complementary V or Vu domains, respectively, see, for example, Portolano et a /. , J. Inmunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
[00153] [00153] Among the antibodies included in the supplied CARs are antibody fragments. An "antibody fragment" or "antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, however, are not limited to, Fv, Fab, Fab ', Fab'-SH, F (ab') 2; diabodies; linear antibodies; variable heavy chain (Vx) regions, single chain antibody molecules such as scFvs and single domain antibodies, comprising only the Vu region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding domain in the provided CARs is or comprises an antibody fragment comprising a variable region of heavy chain (Vn) and a variable of light chain (V.). In particular embodiments, antibodies are single chain antibody fragments comprising a variable region of the heavy chain (Vx) and / or a variable region of the light chain (V.), such as scFvs.
[00154] [00154] Single domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the variable region of the heavy chain or all or a portion of the variable region of the light chain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody.
[00155] [00155] Antibody fragments can be produced by various techniques, including, among others, proteolytic digestion of an intact antibody, as well as production by recombinant host cells. In some embodiments, antibodies are fragments produced recombinantly, as fragments that comprise arrangements that do not occur naturally, such as those with two or more regions or chains of antibodies joined by synthetic ligands, for example, peptide ligands and / or that may not be produced by enzymatic digestion of an intact, naturally occurring antibody. In some respects, the antibody fragments are scFvs.
[00156] [00156] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody can optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity for humans, while maintaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (for example, the antibody from which the CDR residues are derived), for example, to restore or improve specificity or antibody affinity.
[00157] [00157] Among the anti-BCMA antibodies included in the CARs provided
[00158] [00158] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to the antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extracromosomally or are randomly integrated into the animal's chromosomes. In these transgenic animals, the endogenous immunoglobulin loci were generally inactivated. Human antibodies can also be derived from human antibody libraries, including cell-free and phage display libraries, containing antibody coding sequences derived from a human repertoire.
[00159] [00159] Among the antibodies included in the supplied CARs are those that are monoclonal antibodies, including fragments of monoclonal antibodies. The term "monoclonal antibody", when used herein, refers to an antibody obtained from or within a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical, except for possible variants that contain mutations naturally occurring or arising during the production of a monoclonal antibody preparation, these variants being generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. The term should not be interpreted as requiring the production of the antibody by any particular method. A monoclonal antibody can be produced by a variety of techniques, including, but not limited to, generation from a hybridoma, DNA matching methods, phage display and other antibody display methods.
[00160] [00160] In some embodiments, the CAR includes a BCMA-binding portion or parts of the antibody molecule, such as a heavy chain variable region (Vrx) and / or light chain variable region (V.) of the antibody, for example, a scFv antibody fragment. In some embodiments, the supplied BCMA-binding CARs contain an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment that confers the BCMA-binding properties of the supplied CAR. In some embodiments, the antibody or antigen-binding domain can be any anti-BCMA antibody described or derived from any anti-BCMA antibody described. See, for example, Carpenter et a., Clin Cancer Res., 2013, 19 (8): 2048-2060, WO 2016090320, WO 2016090327, WO2010104949 and WO 2017173256. Any of these anti-BCMA antibodies or antigen-binding fragments can be used in the CARs provided. In some embodiments, the anti-BCMA CAR contains an antigen binding domain which is a scFv containing a weighted variable (VH) and / or variable variable (V.) region derived from an antibody described in WO 2016090320 or WO2016090327.
[00161] [00161] In some embodiments, the antibody, for example, the anti-BCMA antibody or antigen-binding fragment, contains a variable region sequence of the heavy and / or light chain (Vr or V1), as described, or a sufficient amount of antigen binding. In some embodiments, the anti-BCMA antibody, for example, antigen binding fragment, contains a sequence from the Vs region or a sufficient antigen binding portion that contains a CDR-H1, CDR-H2 and / or CDR-H3, as described. In some embodiments, the anti-BCMA antibody, for example, antigen binding fragment, contains a V region sequence or sufficient antigen binding portion that contains a CDR-L1, CDR-L2 and / or CDR-L3, as described. In some embodiments, the anti-BCMA antibody, for example, antigen binding fragment, contains a sequence from the Vn region that contains a CDR-H1, CDR-H2 and / or CDR-H3 as described and contains a sequence from the V region which contains a CDR-L1, CDR-L2 and / or CDR-L3 as described. Also among antibodies are those that have sequences of at least or about 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98% or 98%, or about 99% identical to that sequence.
[00162] [00162] In some embodiments, the antibody, for example, its antigen-binding fragment, in the CAR provided, has a heavy chain variable region (Vx) having the amino acid sequence selected from any of the SEQ ID NOs : 1110-115, 247- 256, 324, 325, 518-531, 533, 609, 617 and 772-774 and 814-832, or an amino acid sequence that is at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the Vx region amino acid selected from any of SEQ ID NOs: 110-115, 247-256 324, 325 , 518-531, 533, 609, 617, 772-7T74 and 814-832, or contains a CDR-H1, CDR-H2 and / or CDR-H3 present in that Vx sequence. In some embodiments, the antibody
[00163] [00163] In some embodiments, the Vu region of the anti-BCMA antibody is one that includes a region for determining the complementarity of heavy chain 3 (CDR-H3) that comprises the sequence of amino acids X: X2X3X4aXsXeX7XgXaX10X11X12X13X14 (SEQ ID NO: 355), where X; is A, D, E, G, L, Vou W; X2 is A, D, G, L, P, Q or S; Xgé A, D, G, Lou Y; x is D, G, P, R, S, V, Y or null; Xxé D, I, P, S, T, Y or null; Xe is A, G, 1, S, T, V, Y or null; Xxx is A, DE, FE, L P, S, Y or null; X; is P, Q, T, Y or null; Xs is D, G, R, Y or null; Xin is A, F, Y or null; X11 is D, F or null; X12 is F or null; X13 is D, Tou Y; and Xi él, L, N, Vou Y. In some of these modalities, in said CDR-H3, NMéVv.XxéDXAéG XMéD; XséY; XséV; XréD; Xsé null; Xo is null; X10 is null; X11 is null; X12 is null; XK13 is D; and Km is Y.
[00164] [00164] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H3 that comprises the amino acid sequence selected from any of SEQ ID NOs: 7-11, 149-157, 279- 287, 292, 293, 376-378, 517, 595, according to the Kabat numbering. In some embodiments, the Vu region of an antibody or its antigen-binding fragment contains a CDR-H3 with the amino acid sequence that comprises the amino acid sequence selected from any of SEQ ID NOs: 7-11, 149-157, 279- 287, 292, 293, 376-378, 517 and 595, according to the Chothia numbering or the ADM numbering. In some embodiments, the Vx region of an antibody or antigen-binding fragment contains a CDR-H3 with the amino acid sequence that comprises the amino acid sequence selected from SEQ ID NOs: 606 and 613. In some embodiments, the antibody or antigen- its binding fragment contains a CDR-H3 with the amino acid sequence of SEQ ID NO: 517, 595, 606 or
[00165] [00165] In some embodiments, the Vx region of an antibody or antigen-binding fragment thereof comprises a CDR-H3 contained in the amino acid sequence of the Vx region selected from any of SEQ ID NOs: 110-115, 247 -256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832.
[00166] [00166] In some embodiments, the Vu region of the antibody or its antigen-binding fragment is one that includes a heavy chain 1 complementarity determining region (CDR-H1) that comprises the amino acid sequence of X1X2X3MX, ( SEQ ID NO: 353) XKr is Dou S; Xcé You S; XIg is A, G, Wou Y; and Ka É H / QousS. In some embodiments, in said CDR-H1, 4 is D IC is Y; I3 is Y; eXés.
[00167] [00167] In some embodiments, the Vx region of an antibody or its antigen-binding fragment contains a CDR-H1 with the amino acid sequence that comprises the amino acid sequence selected from any of SEQ ID NOs: 1 -3, 140-144, 288, 289, 507 and 593 according to the Kabat numbering. In some
[00168] [00168] In some embodiments, the Vu region of an antibody or its antigen-binding fragment contains a CDR-H1 contained in the amino acid sequence of the Vn region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832.
[00169] [00169] In some embodiments, the Vx region of an antibody or its antigen-binding fragment is one that includes a heavy chain 2 complementarity determining region (CDR-H2) comprising the KilX2X3aXaXsXeXK7X8XK9X10X11Y X12 X13 X14 amino acid sequence X15 X16 X17 (SEQ ID NO: 354), where X :; is F, G, H, V, Wou Y; Cc is NR, SouV; XA;: is P, Q, S, V, W or Y; X1a is K or null; X5 is A or null; Xs is D, G, N, Sou Y; Xxé GouS; Xsé GouS; XséE, G, N, TouS; XnélKouT; X1éE, G, NouY; Xi is AouV; X3 is A DouQ; XméKousS; X15 is FouV; Xi is KoudQ; and X17 is E or G. In some embodiments in said CDR-H2, XKx is Y; X2ÉéS, X3éS; X1a is null; Xs is null; Xe is S; Xr is G; Xg is S; X9 is T; X1o is | Xi is Y; XR is A; X13 is D; Xm is S; Kis É V: Kis é Ke XiréG.
[00170] [00170] In some embodiments, the Vu region of an antibody or its antigen-binding fragment contains a CDR-H2 that comprises the amino acid sequence selected from any of SEQ ID NOs: 4-6, 145- 148, 290, 291, 372-374, 513 and 594 according to the Kabat numbering. In some embodiments, the Vu region of an antibody or its antigen-binding fragment contains a CDR-H2 that comprises the amino acid sequence selected from any of SEQ ID NOs: 16-18, 161 -164, 296, 297, 514-516, 551, 597, according to Chothia's numbering. In some embodiments, the Vu region of an antibody or its antigen-binding fragment contains a CDR-H2 that comprises the amino acid sequence selected from any of SEQ ID NOs: 23-25, 170-173, 300 , 301, 510-512, 587 and 599 according to the numbering of APM. In some embodiments, the Vn region of an antibody or antigen-binding fragment contains a CDR-H2 that comprises the amino acid sequence selected from any of SEQ ID NOs: 605 and 612. In some modalities, the region An antibody Vn or its antigen binding fragment contains a CDR-H2 with the amino acid sequence of any of SEQ ID NOs: 513, 551, 587, 594, 597, 599, 605 or
[00171] [00171] In some embodiments, the Vx region of an antibody or its antigen-binding fragment contains a CDR-H2 contained in the amino acid sequence of the Vnx region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832.
[00172] [00172] In some embodiments, the antibody or its antigen binding fragment contains a CDR-H1 that is or comprises the amino acid sequence selected from any of SEQ ID NOs: 1-3, 140-144, 288, 289, 507 and 593 according to the Kabat numbering; a CDR-H2 that is or comprises the selected amino acid sequence from any of SEQ ID NOs: 4-6, 145-148,
[00173] [00173] In some embodiments, the Vu region of an antibody or its antigen-binding fragment comprises a CDR-H1, CDR-H2 and / or CDR-H3 according to the Kabat numbering. In some embodiments, the V4 region of an antibody or its antigen-binding fragment comprises a CDR-H1, CDR-H2 and / or CDR-H3 according to the Chothia numbering. In some embodiments, the Vx region of an antibody or its antigen-binding fragment comprises a CDR-H1, CDR-H2 and / or CDR-H3 according to the AbM numbering.
[00174] [00174] In some embodiments, the antibody or its antigen-binding fragment comprises a Vx region comprising a CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of: CDR-H1, CDR-H2 and CDR- H3 comprising the amino acid sequence of SEQ ID NOs: 1, 4 and 7, respectively; CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5 and 8, respectively; CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5 € 9, respectively; CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5 and 10, respectively; CDR-H1, CDR-H2 and CDR-H3 which comprise the amino acid sequence of SEQ ID NOs: 3, 6 and 11, respectively; CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 140, 145 and 149, respectively; CDR-H1, CDR-H2 and CDR-H3 which comprise the amino acid sequence of SEQ ID NOs: 141, 145 and 149, respectively; CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 141, 145 and 150, respectively; CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOs: 142, 146 and
[00175] [00175] For example, the antibody or its antigen-binding fragment provided herein comprises a Vx region comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence selected from: SEQ ID NOs: 1, 4 and 7; SEQID NOs: 2, 5 and 8; SEQ ID NOs: 2, 5 and 9; SEQ ID NOs: 2, 56 10; SEQ ID NOs: 3.6 and 11; SEQ ID NOs: 140, 145 and 149; SEQ ID NOs: 141, 145 and 149; SEQ ID NOs: 141, 145 and 150; SEQ ID NOs: 142, 146 and 151; SEQ ID NOs: 2, and 152; SEQ ID NOs: 143, 147 and 153; SEQ ID NOs: 144148 and 154; SEQ ID NOs: 3, 6 and 155; SEQ ID NOs: 2, 5 and 156; SEQ ID NOs: 2, 5 and 157; SEQ ID NOs: 2, 6 and 376; SEQ ID NOs: 3, 372 and 376; SEQ ID NOs: 3, 6 and 376; SEQ ID NOs: 3, 6 and 377; SEQ ID NOs: 2, 373 and 152; SEQ ID NOs: 2, 5 and 378; SEQ ID NOs: 2, 374 and 9, SEQ ID NOs: 288, 290 and 292; SEQ ID NOs: 289, 291, 293; SEQ ID NOs: 507, 513 and 517; and SEQ ID NOs: 593, 594 and 595, respectively, according to the Kabat numbering.
[00176] [00176] In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, which comprises the amino acid sequence of a CDR-H1, a CDR-H2 and a CDR-H3 contained within the amino acid sequence of the Vx region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832. In some embodiments, the antibody or its antigen-binding fragment comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, which comprises the amino acid sequence of a CDR-H1, a CDR-H2 and a CDR-H3 contained within the amino acid sequence of the Va region of SEQ ID NO: 609 or SEQ IDNO: 617. In some embodiments, the antibody or its antigen-binding fragment comprises a Vx region comprising a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively; SEQ ID NOS: 596, 597 and 595, respectively; SEQ ID NOS: 598, 599 and 595, respectively; or SEQ ID NOS: 611, 612 and 613, respectively.
[00177] [00177] In some embodiments of the antibody or antigen binding fragment provided herein, the Vu region comprises any of the CDR-H1, CDR-H2 and CDR-H3, as described and comprises a region of structure 1 ( FR1), an FR2, an FR3 and / or an FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively for an FR1, an FR2, an FR3 and / or an FR4 contained in the amino acid sequence of the Vn region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531 , 533, 609, 617, 772-77A4, and 814-832. For example, the anti-BCMA antibody or its antigen-binding fragment may comprise a CDR-H1, CDR-H2 and CDR-H3, respectively, contained in the amino acid sequence of the Vn region selected from any SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832 and a frame region (for example, a FR1, a FR2, a FR3 and / or an FR4) containing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity for a region of structure (for example, a FR1, a FR2, a FR3 and / or a FR4) contained in the amino acid sequence of the Vu region selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-
[00178] [00178] In some embodiments, the antibody or its antigen binding fragment comprises a Vx region comprising the amino acid sequence selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518 -531, 533, 609, 617, 772-774 and 814-832.
[00179] [00179] Antibodies and their antigen-binding fragments are also provided, with sequences at least equal to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98% or 99% identical for such sequences. For example, an antibody or antigen binding fragment is provided herein, comprising a Vn region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity with an amino acid sequence of the Vn region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774 and 814-832.
[00180] [00180] In some embodiments, the antibody is a single domain antibody (sdAb) comprising only one sequence from the Vx region or a sufficient antigen-binding portion, like any of the Vx sequences described above (for example, a CDR -H1, a CDR-H2, a CDR-H3 and / or a CDR-H4).
[00181] [00181] In some embodiments, an antibody provided herein (for example, an anti-BCMA antibody) or its antigen-binding fragment, comprising a Vx region further comprises a light chain or a sufficient antigen-binding portion. For example, in some embodiments, the antibody or its antigen-binding fragment contains a Vx region and a V region, or a sufficient antigen-binding portion of a Vx and V region. embodiments, a sequence of the Vx region can be any of the Vx sequences described above. In some of these modalities, the antibody is an antigen-binding fragment, such as a Fab or scFv. In some of these embodiments, the antibody is a complete antibody that also contains a constant region.
[00182] [00182] In some embodiments, the antibody, for example, its antigen-binding fragment, has a variable region of the light chain (V.) with the amino acid sequence selected from any of SEQ ID NOs: 116 -127, 257-267, 326, 327, 534-550, 552-557, 610,618, 775-777 and 833-849, or an amino acid sequence that has at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with a V region amino acid sequence selected from any of SEQ ID NOs: 116-127, 257-267 , 326, 327, 534-550, 552-557, 610,618, 775-777 and 833-849. In some embodiments, the antibody or antigen-binding fragment has a V region described in any of WO 2016090327, WO 2016090320 or WO 2017173256
[00183] [00183] In some embodiments, the Vi region of the antibody described here (for example, an anti-BCMA antibody) or its antigen binding fragment is one that includes a region for determining the complementarity of light chain 3 (CDR-L3) comprising the amino acid sequence X X2X3XaXsXeX7X8XaX10X11X12, (SEQ ID NO: 358), where X: Is A, C, G, H, 1, Qor S; X2 is A, Q, I am V; X3 is S, W or Y; Xa is D, F, G, Hou Y; Xxé D, G, M, R Sou T; Xeé A, G, H, L, R, S, TouY; Xr is L, P, R, S or null; Xs is D, G, N, R, S, T or null; Xs is A, G, H, L, P or null; X1o is EF, S or null; X17 is L, P, Wou Y; and Xi is S, T or V. In some embodiments, in said CDR-L3, X: is H; Xo is VV; Xs is W, Xa is D; KG IS R; Kg IS S; XKr is R; X8a is D; X9 is H; X10 is null; XnéY; eXpéV.
[00184] [00184] In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L3 that comprises the amino acid sequence selected from any of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to the numbering of Kabat, the numbering of Chothia or the numbering of ADM. In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L3 with the amino acid sequence of SEQ ID NO: 591 or 603 according to Kabat numbering, Chothia numbering or numbering of ADM. In any of these examples, the antibody or its antigen-binding fragment may contain a sequence from the V. region selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534- 550, 552-557, 610, 618, 775-777 and 833-849, where the corresponding CDR-L3 sequence contained therein (for example, corresponding to amino acid residues L89 to L97 by Kabat numbering) is replaced by the CDR-L3 sequence selected from any of SEQ ID NOs: 47-58, 184-194, 306, 307, 415- 427, 429-433, 591 and 603 according to Kabat numbering, the number of Chothia or the numbering of AbM.
[00185] [00185] In some modalities, the Vi region. of an antibody or its antigen-binding fragment comprises a CDR-L3 contained in the amino acid sequence of region V. selected from any of SEQ ID NOs: 1116-127, 257-267, 326, 327, 534 -550, 552-557, 610,618, 775-777 and 833-849. In some modalities, the Vi region. An antibody or antigen-binding fragment thereof comprises a CDR-L3 contained in the amino acid sequence of region V. of SEQ ID NO: 610 or SEQ ID NO: 618.
[00186] [00186] In some embodiments, the Vi region of the antibody described here (for example, an anti-BCMA antibody) or its antigen binding fragment is one that includes a light chain complementarity determining region 1 (CDR-L1) which contains the sequence of amino acids: KiX2XK3XaXsXKeX7XgXgX 10X 11X12X13X14X15X16X17 (SEQ ID NO: 356), where X; is G, K RS ouT; X2 is A, GouS; X3éG, N, SouT; XéG, K, N, Q, RouS; XséS or null; Xs is D, N, V or null; X; is L, V or null; Xs is H, S, Y or null; Xs is S, T or null; X1o is S or null; X1: is D, G, |, N, S or null; X12 is D, E, G, K, |, N or null; X13 is FE, GG KN, R, S, Y or null; X11 is D, K, N, T or null; X15 is A, D, G, L, N, S, T or Y; Xisé Lou V; X1i7 is A, H, N, Q or S. In some modalities, X: is G; Xcé A; X3 is N, X4a is N; Xs is null; Xs is null; X; it is null; Xs is null; X9 is null; X1o is null; X11 is |; X12 is G; XK13 IS S; Xu is K X15 IS S; XK16 IS V; XKi7 It's H.
[00187] [00187] In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L1 that comprises the amino acid sequence selected from any of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or ADM numbering. In some embodiments, the antibody or its antigen binding fragment contains a CDR-L1 comprising the selected amino acid sequence from any of SEQ ID NOs: 607 and 614. In some embodiments, the antibody or its binding fragment The antigen contains a CDR-L1 having the amino acid sequence of SEQ ID NO: 589 or 601 according to the numbering of
[00188] [00188] In some modalities, the Vi region. of an antibody or its antigen-binding fragment comprises a CDR-L1 contained in the amino acid sequence of region V. selected from any of SEQ ID NOs: 1116-127, 257-267, 326, 327, 534 -550, 552-557, 610,618, 775-777 and 833-849. In some embodiments, the V. region of an antibody or its antigen-binding fragment comprises a CDR-L1 contained in the amino acid sequence of the V. region of SEQ ID NO: 589, 601, 607 or 614.
[00189] [00189] In some embodiments, the Vi region of the antibody provided here (for example, an anti-BCMA antibody) or its antigen binding fragment is one that includes a light chain 2 complementarity determining region (CDR-L2) containing the amino acid sequence of X1X2X3X4aXsX6X7 (SEQ ID NO: 357), where X: is A, D, E, N, S, Vou W Ico is A, D, NS or V. IG is A, D / H, |, NouS; X: is DK N, Q, RouT; XséL, RouV; Xçé A, E PouQeXréA, D, S or T. In some modalities, X: is D ;: X2c is D Xg is D Xa Is D; Xs is R; X is P; jeXréS.
[00190] [00190] In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L2 that comprises the sequence
[00191] [00191] In some embodiments, the Vi region of an antibody or its antigen-binding fragment comprises a CDR-L2 contained in the amino acid sequence of region V. selected from any of SEQ ID NOs: 1116-127 , 257-267, 326, 327, 534-550, 552-557, 610,618, 775-777 and 833-849. In some modalities, the Vi region. An antibody or its antigen-binding fragment comprises a CDR-L2 contained in the amino acid sequence of region V. of SEQ ID NO: 589, 601, 607 or 614.
[00192] [00192] In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L1 which is or comprises the amino acid sequence selected from any of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or ADM numbering; a CDR-L2 that is or comprises the selected amino acid sequence from any of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409,411-414, 590 and 602 according to the number of Kabat, Chothia numbering or AbM numbering; and a CDR-L3 which is or comprises the selected amino acid sequence from any of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603, in accordance with Kabat numbering, Chothia numbering or AbM numbering.
[00193] [00193] In some embodiments, the Vi region of an antibody or its antigen-binding fragment comprises a CDR-L1, CDR-L2 and / or CDR-L3 according to the Kabat numbering. In some embodiments, the Vi region of an antibody or its antigen-binding fragment comprises a CDR-L1, CDR-L2 and / or CDR-L3 according to the Chothia numbering. In some modalities, the Vi region. An antibody or its antigen-binding fragment comprises a CDR-L1, CDR-L2 and / or CDR-L3 according to the APM numbering.
[00194] [00194] In some modalities of the antibody or its antigen-binding fragment provided here, the V. region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from: CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively; a CDR-L1, CDR-L2 and CDR-L3 which comprises the amino acid sequence of SEQ ID NOs: 28, 39, and 49, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 29, 40, and 50, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 30, 39, and 51, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 31, 41, and 52, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 32, 42, and 53, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 30, 39, and 54, respectively; a CDR-L1, CDR-L2 and CDR-L3 which comprises the amino acid sequence of SEQ ID NOs: 33, 43, and 55, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 34, 44, and 56, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 35, 45, and 57, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 36, 46, and 58, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 174, 179, and 184, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 174, 179, and 185, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprise the amino acid sequence of SEQ ID NOs: 174, 179, and 186, respectively; a CDR-L1, CDR-L2 and CDR-L3 which comprises the amino acid sequence of SEQ ID NOs: 174, 179, and 187, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 175, 180, and 188, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 174, 179, and 189, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID NOs: 176, 181, and 190, respectively; a CDR-L1, CDR-L2 and CDR-L3 that comprises the amino acid sequence of SEQ ID
[00195] [00195] For example, the antibody or its antigen binding fragment provided herein comprises a Vi region that comprises CDR-L1, CDR-L2 and CDR-L3 that comprise the amino acid sequence selected from: SEQ ID NOs: 26 , 37, and 47; SEQ ID NOs: 27, 38, and 48; SEQ ID NOs: 28, 39, and 49; SEQ ID NOs: 29, 40, and 50; SEQ ID NOs: 30, 39, and 51; SEQ ID NOs: 31, 41, and 52; SEQ ID NOs: 32, 42, and 53; SEQ ID NOs: 30, 39, and 54; SEQ ID NOs: 33, 43, and 55; SEQ ID NOs: 34, 44, and 56; SEQ ID NOs: 35, 45, and 57; SEQ ID NOs: 36, 46, and 58; SEQ ID NOs: 174, 179, and 184; SEQ ID NOs: 174, 179, and 185; SEQ ID NOs: 174, 179, and 186; SEQ ID NOs: 174, 179, and 187; SEQ ID NOs: 175, 180, and 188; SEQ ID NOs: 174, 179, and 189; SEQ ID NOs: 176, 181, and 190; SEQ ID NOs: 177, 182, and 191; SEQ ID NOs: 174, 179, and 192; SEQ ID NOs: 178, 183, and 193; SEQ ID NOs: 178, 183, and 194; SEQ ID NOs: 30, 399, and 415; SEQ ID NOs: 380, 400, and 416; SEQ ID NOs: 33, 43, and 421; SEQ ID NOs: 381, 401, and 417; SEQ ID NOs: 382, 402, and 418; SEQ ID NOs: 383, 403, and 419; SEQ ID NOs: 384, 39, and 54; SEQ ID NOs: 385, 180, and 58; SEQ ID NOs: 175, 180, and 188; SEQ ID NOs: 386, 404, and 420; SEQ ID NOs: 387, 405, and 422; SEQ ID NOs: 388, 406, and 423; SEQ ID NOs: 388, 407, and 424; SEQ ID NOs: 389, 408, and 425; SEQ ID NOs: 390, 183 and 193; SEQ ID NOs: 391, 409, and 426; SEQ ID NOs: 392, 40, and 427; SEQ ID NOs: 394, 39, and 429; SEQ ID NOs: 395, 411, and 430; SEQ ID NOs: 396, 412, and 431; SEQ ID NOs: 396, 412, and 58; SEQ ID NOs: 397, 413, and 432; SEQ ID NOs: 398, 414,
[00196] [00196] In some embodiments, the antibody or its antigen-binding fragment contains a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the amino acid sequence of region V. selected from any among SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody contains a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the amino acid sequence of region V. selected from SEQ ID NO: 610 or SEQ ID NO: 618.
[00197] [00197] In some embodiments, the antibody or its antigen-binding fragment comprises the Vi region. which comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602, and 603, respectively; or SEQ ID NOS: 614, 615, and 603, respectively.
[00198] [00198] In some embodiments of the antibody or its antigen binding fragment provided here, the V region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described and comprises a region of structure 1 (FR1I), an FR2, an FR3 and / or an FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity sequence, respectively, to an FR1, an FR2, an FR3 and / or an FRA4 contained within the V region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326 , 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. For example, the anti-BCMA antibody or its antigen-binding fragment may comprise a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the amino acid sequence of region V. selected from any of the SEQs ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610,
[00199] [00199] In some embodiments, the antibody or its antigen-binding fragment comprises the Vi region which comprises an amino acid sequence selected from any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534 -550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody or its antigen-binding fragment contains the V region that comprises
[00200] [00200] Likewise, antibodies have sequences at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical such strings.
[00201] [00201] In some embodiments, the Va region of the antibody or fragment comprises the amino acid sequence selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518- 531, 533, 609, 617, 772-774, and 814-832, and the V. region of the antibody or fragment comprises the amino acid sequence selected from any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849.
[00202] [00202] “Likewise, they are antibodies and their antigen-binding fragments having sequences at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% identical to such strings. For example, an antibody or antigen-binding fragment containing the V region is provided here which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% sequence identity for the V region amino acid sequence selected from any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534- 550, 552-557, 610, 618, 775-777, and 833-849 and / or which comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the Vn region amino acid sequence selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531 , 533, 609, 617, 772-774, and 814-832. In some embodiments, the antigen or antigen-binding fragment contains the Vi region that comprises the amino acid sequence selected from any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534 -550,
[00203] [00203] In some embodiments, the Vi region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vu region sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the Vi region. is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
[00204] [00204] In some embodiments, the Vu region and the V regions comprise the sequence of SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the Vi regions. comprise the sequence of SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence having at least 90% identity for this; the Vr region and the Vi regions comprise the sequence of SEQ ID NOs: 111 and 117, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence having at least 90% identity for this; the Vu region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 119, respectively, or an amino acid sequence having at least 90% identity for this; the VH region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 120, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 121, respectively, or an amino acid sequence having at least 90% identity for this; the Vu region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 122, respectively, or an amino acid sequence having at least 90% identity for this; the Vr region and the Vi regions comprise the sequence of SEQ ID NOs: 110 and 123, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the V region comprise the sequence of SEQ ID NOs: 112 and 124, respectively, or an amino acid sequence having at least 90% identity for this; the Vu region and the V regions comprise the sequence of SEQ ID NOs: 113 and 125, respectively, or an amino acid sequence having at least 90% identity for this; the VH region and the Vi regions comprise the sequence of SEQ ID NOs: 114 and 126, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the V region comprise the sequence of SEQ ID NOs: 115 and 127, respectively, or an amino acid sequence having at least 90% identity for this; the Vu region and the V regions comprise the sequence of SEQ ID NOs: 247 and 257, respectively, or an amino acid sequence having at least 90% identity for this; the Vu region and the V regions comprise the sequence of SEQ ID NOs: 248 and 258, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the V regions comprise the sequence of SEQ ID NOs: 249 and 259, respectively, or an amino acid sequence having at least 90% identity for this; the Vx region and the V regions comprise the sequence of SEQ ID NOs: 250 and 260, respectively, or an amino acid sequence having at least 90% identity for this; the region
[00205] [00205] In some embodiments, the Vu region of the antibody or its antigen-binding fragment comprises a CDR-H1, a CDR-H2, a CDR-H3, respectively, which comprises the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained within the Vx region amino acid sequence selected from any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772 -774, and 814-832; and comprises a CDR-L1, a CDR-L2, a CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3, respectively contained within the amino acid sequence of region V, selected from any of SEQ ID NOs: 618,116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, and 833-849.
[00206] [00206] In some of any embodiments, Vx is or comprises CDR-H1, CDR-H2 and CDR-H3 contained within the Vn sequence of SEQ ID NO: 617; and Vi is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. sequence of SEQ ID NO: 618; Vh is or comprises CDR-H1, CDR-H2 and CDR-H3 contained within the V4 sequence of SEQ ID NO: 256; and Vi is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. sequence of SEQ ID NO: 267; Vu is or comprises CDR-H1, CDR-H2 and CDR-H3 contained within the V4 sequence of SEQ ID NO: 519; and Vi is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. sequence of SEQ ID NO: 535; Vu is or comprises CDR-H1, CDR-H2 and CDR-H3 contained within the Vx sequence of SEQ ID NO: 115; and V. is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V sequence, of SEQ ID NO: 536; or Vy is or comprises CDR-H1, CDR-H2 and CDR-H3 contained within the V4 sequence of SEQ ID NO: 609; and aVL is or comprises CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 610. In some embodiments, the Vn region comprises CDR-H1, CDR-H2 and CDR-H3 contained within of the sequence
[00207] [00207] In some embodiments, the Vx region is or comprises (a) a CDR-H1 comprising the sequence selected from any of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289 , 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 comprising the sequence selected from any of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 comprising the sequence selected from any of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517, or 606; and the V. region is or comprises (a)
[00208] [00208] In some embodiments, the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 601, 602, and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 1,4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the sequence of SEQ ID NOS: 26, 37, and 47, respectively; the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 2, 5, and 8, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the sequence of SEQ ID NOS: 27, 38, and 48, respectively; the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 1, 4, and 7, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the sequence of SEQ ID NOS: 28, 39, and 49, respectively; the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 1, 4, and 7, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the sequence of SEQ ID NOS: 29, 40, and 50, respectively; the V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of
[00209] [00209] In some embodiments, the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 596, 597, and 595, respectively, and the V. region comprises a CDR-L1 , CDR-L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 601, 602, and 603, respectively. In some modalities, the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of SEQ ID NOS: 598, 599, and 595, respectively, and the VL region comprises a CDR-L1 , CDR-L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 601, 602, and 603, respectively. In some modalities, the Vx region comprises
[00210] [00210] In some embodiments, the Vx region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814 -832; and the V. region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
[00211] [00211] In some embodiments, the V4 and V. regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 116, respectively; the Vx and V. regions of the antibody or its antigen binding fragment comprise the amino acid sequences of SEQ ID NOs: 111 and 117, respectively; the Vn and V regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 118, respectively; the Vx and V. regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 119, respectively; the Vu and V. regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 120, respectively; the Vu and Vi regions. the antibody or its antigen-binding fragment comprises the amino acid sequences of SEQ ID NOs: 110 and 121, respectively; the Vn and V. regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 122, respectively; the Vn and V. regions of the antibody or its antigen-binding fragment comprise the amino acid sequences of SEQ ID NOs: 110 and 123, respectively; the Vu and V. regions of the antibody or its antigen-binding fragment comprise the following
[00212] [00212] For example, the Vu and Vi regions. the antibody or its antigen-binding fragment provided in these comprise the amino acid sequences selected from: SEQ ID NOs: 110 and 116; SEQ ID NOs: 111 and 117; SEQ ID NOs: 110 and 118; SEQ ID NOs: 110 and 119; SEQ ID NOs: 110 and 120; SEQ ID NOs: 110 and 121; SEQ ID NOs: 110 and 122; SEQ ID NOs: 110 and 123; SEQ ID NOs: 112 and 124; SEQ ID NOs: 113 and 125; SEQ ID NOs: 114 and 126; SEQ ID NOs: 115 and 127; SEQ ID NOs: 247 and 257; SEQ ID NOs: 248 and 258; SEQ ID NOs: 249 and 259; SEQ ID NOs: 250 and 260; SEQ ID NOs: 251 and 261; SEQ ID NOs: 252 and 262; SEQ ID NOs: 253 and 263; SEQ ID NOs: 254 and 264; SEQ ID NOs: 255 and 265; SEQ ID NOs: 256 and 266; SEQ ID NOs: 256 and 267; SEQ ID NOs: 518 and 534; SEQ ID NOs: 519 and 535; SEQ ID NOs: 115 and 536; SEQ ID NOs: 520 and 264; SEQ ID NOs: 521 and 537; SEQ ID NOs: 522 and 538; SEQ ID NOs: 523 and 539; SEQ ID NOs: 519 and 540; SEQ ID NOs: 524 and 541; SEQ ID NOs: 525 and 261; SEQ ID NOs: 526 and 542; SEQ ID NOs: 527 and 543; SEQ ID NOs: 528 and 544; SEQ ID NOs: 529 and 545; SEQ ID NOs: 528 and 546; SEQ ID NOs: 522 and 547; SEQ ID NOs: 256 and 548; SEQ ID NOs: 530 and 549; SEQ ID NOs: 531 and 550; SEQ ID NOs: 519 and 552; SEQ ID NOs: 110 and 553; SEQ ID NOs: 110 and
[00213] [00213] In some embodiments, the Vx and V. regions of the antibody or its antigen-binding fragment provided in these comprise the amino acid sequences selected from: SEQ ID NOs: 617 and 618; SEQ ID NOs: 256 and 267; SEQ ID NOs: 519 and 535; SEQ ID NOs: 115 and 536; or SEQ ID NOs: 609 and 610; respectively, or any antibody or its antigen-binding fragment that has at least 90% sequence identity to any of the Vmx and VL above, such as at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% sequence identity for this, or any antibody or antigen-binding fragment that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the region
[00214] [00214] In some embodiments, the Vu and Vi regions of the antibody or its antigen-binding fragment provided in these comprise the amino acid sequences selected from: SEQ ID NOs: 617 and 618, or any antibody or fragment thereof antigen binding that has at least 90% sequence identity to any of the Vy and VL above, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity for this, or any antibody or antigen-binding fragment comprising CDR-H1, CDR-H2 and CDR-H3 contained within the Vu region, and a CDR-L1, CDR- L2 and CDR-L3 contained within region V. of any of the Vx and V. above.
[00215] [00215] In some embodiments, the antibody or its antigen-binding fragment is a single chain antibody fragment, such as a single chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the Vx region. In some embodiments, the antibody or antigen-binding fragment is an scFv comprising a variable region of the chain heavy (Vx) and a variable region of the light chain (V.). In some embodiments, the single chain antibody fragment (for example, scFv) includes one or more ligands that join two antibody domains or regions, such as a heavy chain variable region (Vx) and a light chain variable region (V.). The linker is typically a peptide linker, for example, a flexible and / or soluble peptide linker. Among the binders are those rich in glycine and serine and / or in some cases threonine. In some embodiments, the binders also include charged residues, such as lysine and / or glutamate, which can improve solubility. In some embodiments, the binders also include one or more prolines.
[00216] Accordingly, the anti-BCMA antibodies provided include single-chain antibody fragments, such as scFvs and diabody, particularly human single-chain antibody fragments, typically comprising linker (s) that unite two antibody domains or regions, such as Vu and V regions. The linker is typically a peptide linker, for example, a flexible and / or soluble peptide linker, such as one rich in glycine and serine.
[00217] [00217] In some respects, binders rich in glycine and serine (and / or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of these amino acid (s). In some modalities, they include at least about 50%, 55%, 60%, 70% or 75% glycine, serine and / or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine and / or threonine. The linkers are generally between about 5 and about 50 amino acids in length, typically between a or about 10 and a or about 30, for example, 10, 11, 12,13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 and in some examples between 10 and 25 amino acids in length. Exemplary ligands include ligands with various numbers of repetitions of the sequence GGGGS (4GS; SEQ ID NO: 359) or GGGS (3GS; SEQ ID NO: 360), as well as between 2, 3, 4 and 5 repetitions of that sequence. Exemplary linkers include those that have or consist of a sequence mentioned in SEQ ID NO: 361 (GGGGSGGGGSGCGGGGS). Exemplary linkers also include those that have or consist of the sequence mentioned in SEQ ID NO: 362 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those that have or consist of the sequence mentioned in SEQ ID NO: 778 (SRGGGGSGGGGSGGGGSLEMA).
[00218] [00218] Therefore, in some modalities, the modalities
[00219] [00219] In some embodiments, the linker has an amino acid sequence containing the sequence mentioned in SEQ ID NO: 361. The fragment, for example, scFv, can include a Vx region or a portion thereof, followed by the linker, followed by a Vi region. or a portion of it. The fragment, for example, scFv, can include the V region or part of it, followed by the ligand, followed by the Vn region or part of it.
[00220] [00220] In some modalities, the antigen-binding domain comprises the sequence selected from any one of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578- 583 , 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the sequence selected from any of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585, or 769-771.
[00221] [00221] In some respects, a scFv provided herein comprises the amino acid sequence selected from any of SEQ ID NOs: 128-139, 268-278, 328, 329, 442, 478, 558-576, 578- 583 , 585, 586, and 769-771, or have an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the selected amino acid sequence from any one of SEQ ID NOs: 128-139, 268-278, 328, 329, 442, 478, 558-576, 578-583, 585, 586 , and 769-771.
[00222] [00222] For example, the scFv provided here comprises the sequence
[00223] [00223] Table 2 provides SEQ ID NOS: of exemplary antigen-binding domains, such as antibodies or antigen-binding fragments, which may be comprised in the supplied BCMA-binding receptors, such as anti-chimeric antigen receptors (CARs) -BCMA. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or a fragment thereof, comprising a Vx region comprising the sequence of CDR-H1, CDR-H2 and CDR-H3 and a Vi region that comprises the sequence of CDR-L1, CDR-L2 and CDR-L3 mentioned in SEQ ID NOS: listed in each row of Table 2 below (by Kabat numbering). In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or a fragment thereof, comprising a sequence from the Vx region and a sequence from the V region.
[00224] [00224] Among the antibodies, for example, antigen-binding fragments, in the CARs provided, are human antibodies. In some embodiments of a human anti-BCMA antibody provided, for example, antigen binding fragments, the human antibody contains a Vs region comprising a portion of at least 95%, 96%, 97%, 98%, 99 % or 100% sequence identity with an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence encoded by a germline nucleotide human heavy chain D segment and / or a portion of at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity for an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and / or contains a V region that comprises a portion with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence encoded by a V chain segment germline nucleotide layer or human lambda and / or a portion with at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence encoded by a J segment of ca - there is a human germline nucleotide layer or lambda chain. In some modalities, the portion of the Vx region corresponds to the
[00225] [00225] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a CDR-H1 with at least 95%, 96%, 97%, 98%, 99% or 100% identity sequence with an amino acid sequence from the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H1 with a 100% identical sequence or with no more than one, two or three amino acid differences compared to the corresponding CDR-H1 region within a coded sequence by a germline nucleotide human heavy chain V segment.
[00226] [00226] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a CDR-H2 with at least 95%, 96%, 97%, 98%, 99% or 100% identity sequence with an amino acid sequence from the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H2 having a sequence that is 100% identical or with no more than one, two or three amino acid differences compared to the corresponding CDR-H2 region within a coded sequence by a germline nucleotide human heavy chain V segment.
[00227] [00227] In some embodiments, the human antibody, for example
[00228] [00228] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a CDR-L1 with at least 95%, 96%, 97%, 98%, 99% or 100% identity sequence with an amino acid sequence from the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L1 with a 100% identical sequence or with no more than one, two or three amino acid differences compared to the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment.
[00229] [00229] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a CDR-L2 with at least 95%, 96%, 97%, 98%, 99% or 100% identity sequence with an amino acid sequence from the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L2 with a 100% identical sequence or with no more than one, two or three amino acid differences compared to the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment.
[00230] [00230] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a CDR-L3 with at least 95%, 96%, 97%, 98%, 99% or 100% identity sequence with an amino acid sequence from the corresponding CDR-L3 region within a sequence encoded by segment V and segment J of human germline nucleotide light chain. For example, the human antibody in some embodiments contains a CDR-L3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences compared to the corresponding CDR-L3 region within of a sequence encoded by segment V and segment J of human germline nucleotide light chain.
[00231] [00231] In some embodiments, the human antibody, for example, antigen-binding fragment, contains a region of structure that contains sequences of human germline gene segments. For example, in some embodiments, the human antibody contains a Vn region in which the framework region, for example FR1, FR2, FR3 and FRA4, is at least 95%, 96%, 97%, 98%, 99% or 100 % sequence identity for a structure region encoded by a human germline antibody segment, such as a V and / or J segment. In some embodiments, the human antibody contains a V region. which structure region p. FR1, FR2, FR3 and FRA4, have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity for a structure region encoded by a human germline antibody segment, co-
[00232] [00232] In some embodiments, the reference antibody may be an anti-BCMA scFv described in International Patent Application Pub No. WO 2010/104949.
[00233] [00233] The antibody, for example, antigen-binding fragment, can contain at least a portion of an immunoglobulin constant region, such as one or more domain of the constant region. In some embodiments, the constant regions include a light chain constant region and / or a heavy chain 1 (CH1) constant region. In some embodiments, the antibody includes a CH2 and / or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
[00234] [00234] In some embodiments, the recombinant receptor, such as a CAR comprising an antibody (e.g., antigen binding fragment) provided herein, further includes a spacer or spacer region. The spacer is typically a polypeptide spacer and, in general, is located within the CAR between the antigen-binding domain and the transmembrane domain of the CAR. In some respects, the spacer may be or include at least a portion of an immunoglobulin constant region or its modified variant or version, such as an immunoglobulin hinge region, such as an IgG hinge region, for example, an derived from IgG4 or IgG4 region of the joint and / or a region of CH1 / CL and / or Fc. In some embodiments, the constant region or one or more parts of it is a human IgG, such as a human IgG4 or IgG1 or IgG2. In general, the spacer, like the portion of the constant region, serves as a spacer region between the antigen recognition component (for example, scFv), and the transmembrane domain. In some modalities, the length and / or composition of the spacer is modified to optimize or promote certain characteristics of the interaction between the CAR and its target; in some aspects, it is modified to optimize the biophysical synapsed distance between the cell that expresses the CAR, and the cell that expresses the CAR target during or after or after binding the CAR to its target in the cell that expresses the target; in some ways, the cell that expresses the target is a tumor cell that expresses BCMA. In some embodiments, the CAR is expressed by a T cell, and the length of the spacer is compatible with the activation of the T cell or to optimize the performance of the T cell of the CAR. In some embodiments, the spacer is a spacer region, located between the ligand-binding domain and the transmembrane domain of the recombinant receptor, for example, CAR. In some embodiments, the spacer region is a region located between the ligand-binding domain and the transmembrane domain of the recombinant receptor, for example, CAR.
[00235] [00235] In some embodiments, the spacer may have a length that provides greater responsiveness of the cell after binding to the antigen, compared to the absence of the spacer and / or in the presence of a different spacer, such as a different spacer. - in length. In some embodiments, the spacer has at least 100 amino acids in length, such as at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 180, 190, 200, 210, 220, 230 , 240 or 250 amino acids in length. In some examples, the spacer is about 12 amino acids in length or no more than 12 amino acids in length. Exemplary spacers include those that have at least about 10 to 300 amino acids, about 10 to 200 amino acids, about 50 to 175 amino acids, about 50 to 150 amino acids, about 10 to 125 amino acids, about 50 to 100 amino acids, about 100 to 300 amino acids, about 100 to 250 amino acids, about 125 to 250 amino acids or about 200 to 250 amino acids, and including any integer between the end points of any of the listed ranges. In some modes, the spacer or spacer region is at least about 12 amino acids, at least about 119 amino acids or less, at least about 125 amino acids, at least about 200 amino acids, or at least about 220 amino acids, or at least about 225 amino acids in length.
[00236] [00236] In some embodiments, the spacer is 125 to 300 amino acids long, 125 to 250 amino acids long, 125 to 230 amino acids long, 125 to 200 amino acids long, 125 to 180 amino acids long length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some modes, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the above.
[00237] [00237] “Exemplary spacers include those that contain a portion (s) of an immunoglobulin constant region, such as those that contain an Ig joint, such as an IgG joint domain.
[00238] [00238] In some embodiments, the spacer may be derived wholly or partially from IgG4 and / or IgG2 and may contain mutations, such as one or more isolated amino acid mutations in one or more domains. In some instances, amino acid modification is a substitution of a proline (P) for a serine (S) in the hinge region of an IgG4. In some modalities, the modification of amino acids is a substitution of an asparagine (N) for a glutamine (Q) to reduce the heterogeneity of glycosylation, such as an N177Q mutation at position 177, in the Cx2 region, of complete IgG4 Fc sequence mentioned in SEQ ID NO: 750 or in position N176Q.at 176, in the Cr2 region, of the full-size IgG2 Fc sequence defined in SEQ ID NO: 749. In some embodiments, the spacer is or comprises an IgG4 / 2 chimeric joint or modified I9G4 joint; an I9G2 / 4 chimeric Cx2 region; and a CH3 region of IgG4 and optionally has about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649. In some embodiments, the spacer comprises the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVOQFNWYVDGVEVHNAKTKPREEQFOSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ
[00239] [00239] Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19: 3153, Hudecek et al. (2015) Cancer Immunol. Res., 3 (2): 125-135 or publication of the international patent application “number WO 2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce the heterogeneity of RNA after expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce the cryptic binding sites or to reduce the likelihood of a binding event at a binding site.
[00240] [00240] In some embodiments, the spacer has the amino acid sequence mentioned in SEQ ID NO: 363 and is encoded by the polynucleotide sequence mentioned in SEQ ID NO: 364. In some embodiments, the spacer has the amino acid sequence mentioned in SEQ ID NO: 365. In some embodiments, the spacer has the amino acid sequence mentioned in SEQ ID NO: 366. In some embodiments, the spacer has the amino acid sequence mentioned in SEQ ID NO: 630 and is encoded by the polynucleotide sequence mentioned in SEQ ID NO: 629. In some modalities, the spacer has an amino acid sequence established in SEQ ID NO: 649, encoded by the polynucleotide sequence mentioned in SEQ ID NO: 621,622,855 or 856 or one polynucleotide-
[00241] [00241] In some embodiments, the spacer is or comprises an amino acid sequence encoded by the nucleotide sequence mentioned in SEQ ID NO: 622.
[00242] [00242] The antigen recognition component is usually linked to one or more regions of intracellular signaling containing signaling components, such as signaling components that mimic stimulation and / or activation through an antigen receptor complex, as a TCR complex, in the case of a CAR, and / or signal through another cell surface receptor. Thus, in some embodiments, the BOCMA-binding molecule (for example, antibody or antigen-binding fragment thereof) is linked to one or more transmembrane domains, such as those described herein, and intracellular signaling regions or domains comprising one or more intracellular components, such as those described here at. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain is used which is naturally associated with one of the domains on the receiver, for example, CAR. In some cases, the transmembrane domain is selected or modified by substituting amino acids to prevent binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[00243] [00243] The transmembrane domain in some modalities is derived from a natural source or a synthetic source. Where the source is natural, in some respects, the domain is derived from any membrane-bound or transmembrane protein. Trans-membrane domains include those derived (i.e., comprise at least the transmembrane domain (s)) from the alpha, beta or zeta chain of the T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9 , CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 and / or CD154. For example, the transmembrane domain can be a CD28 transmembrane domain comprising the amino acid sequence mentioned in SEQ ID NO: 624, encoded by the nucleic acid sequence mentioned in SEQ ID NO: 623 or SEQ ID NO: 688. Alternatively, the transmembrane domain in some modalities is synthetic. In some respects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some respects, a phenylalanine, tryptophan and valine triplet will be found at each end of a synthetic transmembrane domain. In some modes, the connection is made by ligands, spacers and / or transmembrane domains.
[00244] [00244] Among the regions or domains of intracellular signaling are those that mimic or approximate a signal through a natural antigen receptor, a signal through that receptor in combination with a co-stimulating receptor and / or a signal through a co-stimulating receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of 2 to 10 amino acids in length, such as one containing glycine and serines, for example, glycine-serine doublet, is present and forms a link between the transmembrane domain and the intracellular signaling domain of the CAR.
[00245] [00245] The receiver, for example, the CAR, generally includes an intracellular signaling region comprising at least one intracellular signaling component or components. In some modalities, the receptor includes an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain that mediates T cell activation and cytotoxicity, for example, the CD3 zeta chain. Thus, in some respects, the BCMA-binding antibody is linked to one or more cell signaling modules. In some embodiments, cellular signaling modules include the CD3 transmembrane domain, CD3 intracellular signaling domains and / or other CD transmembrane domains. In some embodiments, the receptor, for example, CAR, further includes a portion of one or more additional molecules, such as the Fc, CD8, CD4, CD25 or CD16 y receptor. For example, in some respects, CAR includes a chimeric molecule between CD3-zeta (CD3-7) or Fc and CD8, CD4, CD25, or CD16 y receptor.
[00246] [00246] In some modalities, after or after CAR binding, the CAR cytoplasmic domain or intracellular signaling domain stimulates and / or activates at least one of the normal effector functions or immune cell responses, for example, T cell modified to express the CAR. For example, in some contexts, CAR induces a T cell function, such as cytolytic activity or T auxiliary activity, such as cytokine secretion or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an immunostimulatory chain.
[00247] [00247] In the context of a natural TCR, complete activation generally requires not only signaling through the TCR, but also a co-stimulating signal. Thus, in some modalities, to promote complete activation, a component to generate a secondary signal or co-stimulator is also included in the CAR. In other modalities, the CAR does not include a component to generate a co-stimulating signal. In some ways, an additional CAR is expressed in the same cell and provides the component to generate the secondary or co-stimulator signal.
[00248] [00248] T cell activation is, in some respects, described as mediated by two classes of cytoplasmic signal sequences: those that initiate primary antigen-dependent activation via TCR (primary cytoplasmic signal sequences) and those that act independently of antigen to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some respects, the CAR includes one or both classes of cytoplasmic signaling sequences.
[00249] [00249] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary stimulation and / or activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulating manner may contain
[00250] [00250] In some embodiments, the CAR includes a signaling domain (for example, an intracellular or cytoplasmic signaling domain) and / or transmembrane portion of a co-stimulating molecule, such as a cell co-stimulating molecule T. Exemplary costimulatory molecules include CD28, 4-1BB, OXA40, DAP10 and ICOS. For example, a costimulatory molecule can be derived from 4-1BB and can comprise the amino acid sequence mentioned in SEQ ID NO: 626, encoded by the nucleotide sequence mentioned in SEQ ID NO: 625 or SEQ ID NO: 681. In some aspects, the same CAR includes the stimulating or activating components (for example, cytoplasmic signaling sequence) and the co-stimulating components.
[00251] [00251] In some modalities, the stimulating or activating components are included within a CAR, while the co-stimulating component is provided by another CAR recognizing another antigen. In some embodiments, CARs include activating or stimulating CARs and co-stimulating CARs, both expressed in the same cell (see, WO2014 / 055668). In some respects, the BCMA-targeted CAR is the stimulating or activating CAR; in other ways
[00252] [00252] In certain embodiments, the intracellular signaling region comprises a CD28 signaling and transmembrane domain linked to an CD3 intracellular domain (e.g., CD3-zeta). In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 co-stimulatory domain (4-1BB, TNFRSFS9), linked to an CD3 zeta intracellular domain.
[00253] [00253] In some modalities, the CAR comprises one or more, for example, two or more co-stimulating domains and a stimulating or activation domain, for example, primary activation domain, in the cytoplasmic portion. Examples of CARs include intracellular components of CD3-zeta, CD28 and 4-1BB.
[00254] [00254] In some embodiments, the chimeric antigen receptor provided comprises: (a) an extracellular antigen binding domain that specifically recognizes B cell maturation antigen (BCMA), like any antigen binding domain described herein; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region. In some modalities, the antigen-binding domain of this receptor comprises a Vx region and a Vi region. which comprises the amino acid sequence of SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence with at least 90% identity to SEQ ID NOS: 617 and 618, respectively. In some embodiments, the antigen-binding domain of that receptor comprises a Vx region that is or comprises CDR-H1, CDR-H2 and CDR-H3 contained in the amino acid sequence of the Vx region of SEQ ID NO: 617 ; and a V. region that is or comprises CDR-L1, CDR-L2 and CDR-L3 contained in the amino acid sequence of region V. of SEQ ID NO: 618. In some modalities, the antigen binding domain of that receptor , comprises a Vx region comprising a CDR-H1, CDR-H2 and CDR-H3 comprising SEQ ID NOS: 593, 594 and 595, respectively, and a V region comprising a CDR-L1, CDR-L2 and CDR-L3 with - comprising SEQ ID NOS: 601, 602 and 603, respectively.
[00255] [00255] Chimeric anticeptors are provided, comprising: (1) an extracellular antigen binding domain that specifically binds to human B cell maturation antigen (BCMA), wherein the extracellular antigen binding domain comprises: ( i) a variable heavy chain (Vs) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the sequence of the Vx region of SEQ ID NO: 617; and (ii) a variable region of the light chain (V.) comprising an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98 % or 99% sequence identity for the V region sequence of any of SEQ ID NO: 618; (2) a spacer mentioned in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence mentioned in SEQ ID NO: 622; (3) a transmembrane domain, optionally a trans-membrane domain of a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta chain (CD36) and an intracellular signaling domain of a T cell co-stimulating molecule. Polynucleotides encoding are also provided. that chimeric antigen receptor.
[00256] [00256] In some embodiments, the Vh region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the Vu region sequence of SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. region sequence of SEQ ID NO: 618; or the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 that comprise the sequence of SEQ ID NOS: 593, 594, and 595, respectively, and the Vi region. comprises CDR-L1, CDR-L2 and CDR-L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively; the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 that comprise the sequence of SEQ ID NOS: 596, 597, and 595, respectively, and the V region comprises CDR-L1, CDR-L2 and CDR -L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively; the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 that comprise the sequence of SEQ ID NOS: 598, 599, and 595, respectively, and the V. region comprises CDR-L1, CDR-L2 and CDR -L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively;
[00257] [00257] Chimeric antigen receptors are provided, comprising: (1) an extracellular antigen-binding domain that specifically binds to the human B-cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises comprises: a heavy chain variable region (Vx) comprising CDR-H1, CDR-H2 and CDR-H3 contained within the Vn region sequence of SEQ ID NO: 617; and the light variable region (V.) comprising CDR-L1, CDR-L2 and CDR-L3 contained within the V. region sequence of SEQ ID NO: 618; or the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the Vx region sequence of SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the V. region sequence of SEQ ID NO: 618; or the Vr region comprises CDR-H1, CDR-H2 and CDR-H3 which comprise the sequence of SEQ ID NOS: 593, 594, and 595, respectively, and the VL region comprises CDR-L1, CDR-L2 and CDR -L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively; the Vmx region comprises CDR-H1, CDR-H2 and CDR-H3 which comprise the sequence of SEQ ID NOS: 596, 597, and 595, respectively, and the VL region comprises CDR-L1, CDR-L2 and CDR- L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively; the VmH region comprises CDR-H1, CDR-H2 and CDR-H3 which comprise the sequence of SEQ ID NOS: 598, 599, and 595, respectively, and the VL region comprises CDR-L1, CDR-L2 and CDR- L3 that comprise the sequence of SEQ ID NOS: 601, 602, and 603, respectively; or the Vu region comprises CDR-H1, CDR-H2 and CDR-H3 which comprise the sequence of SEQ ID NOS: 611, 612, and 613, respectively, and the V region comprises CDR-L1, CDR- L2 and CDR-L3 comprising the sequence of SEQ ID NOS: 614, 615, and 603, respectively; (2) a spacer mentioned in SEQ ID NO: 649 or in which the nucleic acid encoding the spacer is or comprises the sequence mentioned in SEQ ID NO: 622; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a human CD3-zeta chain (CD3C7) and an intracellular signaling domain of a T-cell co-stimulating molecule, optionally from a 4- Human 1BB or a human CD28. Polynucleotides are also provided that complicate this chimeric antigen receptor. In some modalities, the extracellular antigen binding domain comprises the sequence of the Vu region of SEQ ID NO: 617, and the sequence of the V. region of SEQ ID NO: 618. In some modalities, the binding domain to the antigen of such a receptor, which comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, other domains, regions or components of the chimeric antigen receptor include any domains, regions or components described herein at.
[00258] [00258] In some embodiments, the CAR also includes a substitute marker, such as a cell surface marker (for example, a truncated cell surface marker), which can be used to confirm the transduction or modification of the cell to express the re- ceptor. For example, in some respects, extrinsic marker genes are used in connection with cell therapies modified to allow the detection or selection of cells and, in some cases, also to promote cell suicide by ADCC. Examples of marker genes include the truncated epidermal growth factor receptor (EGFRt), which can be co-expressed with a transgene of interest (for example, a CAR or TCR) in transduced cells (see, for example, U.S. Patent No. 8,802,374). EGFRt contains an epitope recognized by the cetuximab antibody (Erbitux & O). For this motif, Erbitux & can be used to identify or select cells that have been modified with the construction of EGFR, including cells also comodified with another recombinant receptor, such as a chimeric antigen (CAR) receptor. In addition, EGFRt is commonly used as a suicide mechanism in connection with cell therapies.
[00259] [00259] “Exemplary substitute markers may include truncated forms of cell surface polypeptides, such as truncated forms that are not functional and do not transduce or are not able to transduce a signal or a signal normally transduced by the complete form of the cell surface polypeptide , and / or are or are not able to internalize.
[00260] [00260] In some modalities, the marker is a selection marker. In some modalities, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some modalities, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that imparts antibiotic resistance to a mammalian cell. In some modalities, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form of it.
[00261] [00261] In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, for example, T2A. See, WO2014031687. In some embodiments, the introduction of a construction encoding the CAR and substitute marker, separated by a T2A ribosome switch, can express two proteins of the same construction, so that the substitute marker can be used as a marker for detect cells that express such a construct. In some embodiments, the substitute marker and, optionally, a binding sequence, can be any as described in international publication no. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) or PSMA that is optionally linked to a linker sequence, such as a 2A cleavable linker sequence (for example, a T2A, P2A, E2A or F2A cleavage linker, described elsewhere in this document) An exemplary polypeptide for a truncated EGFR substitute marker comprises the amino acid sequence mentioned in SEQ ID NO: 634 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of SEQ ID NO: 634 sequence identity. In some embodiments , the spacer is or comprises a sequence rich in glycine-serine or other flexible linker, as known flexible linkers.
[00262] [00262] In some modalities, the marker is a molecule, for example, cell surface protein, not found naturally in T cells or not found naturally on the surface of T cells or in a portion of them.
[00263] [00263] In some modalities, the molecule is a non-own molecule, for example, non-own protein, that is, one that is not recognized as "own" by the host's immune system in which the cells will be transferred adoptively.
[00264] [00264] In some modalities, the marker has no therapeutic function and / or produces another effect in addition to being used as a marker for genetic engineering, for example, to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or molecule that exerts some desired effect, such as a ligand for a cell to be found in vivo, such as an immunostimulatory or immunological checkpoint molecule to improve and / or decrease cell responses after transfection
[00265] [00265] In some cases, CARs are referred to as first, second and / or third generation CARs. In some respects, a first-generation CAR is one that only provides a signal induced by the CD3 chain on or in response to binding to the antigen; in some respects, a second generation CAR is one that provides that signal and co-stimulating signal, such as one that includes an intracellular signaling domain from a co-stimulating receptor such as CD28 or CD137; in some respects, a third generation CAR in some respects is one that includes multiple co-stimulatory domains from different co-stimulatory receptors.
[00266] [00266] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some respects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some modalities, the antibody or fragment includes an scFv or a single domain antibody comprising only the Vu region, and the intracellular signaling domain contains an ITAM. In some respects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta chain (CD367). In some modalities, the chimeric antigen receptor includes a trans-membrane domain linking the extracellular domain, and the intracellular signaling domain. In some respects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and the transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain, and the transmembrane, are connected by a spacer, like any one described here. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a co-stimulating molecule (for example,
[00267] [00267] In some embodiments, the transmembrane domain of the receptor (for example, CAR) is a transmembrane domain of human CD28 or a variant thereof, for example, a 27 amino acid transmembrane domain of a human CD28 (# of access: P10747.1). In some embodiments, the intracellular signaling domain comprises an intracellular co-stimulating signaling domain of human CD28 or its functional variant, such as a 41 amino acid domain and / or a domain with an LL to GG substitution at positions 186- 187 of a native CD28 protein. In some embodiments, the intracellular domain comprises a 4-1BB intracellular co-stimulating signaling domain or a functional variant thereof, such as a 42 amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011,1) . In some modalities, the intracellular signaling domain comprises a stimulating signaling domain of human CD3 zeta or a functional variant thereof, such as a 112 AA cytoplasmic domain of human CD36 isoform 3 (accession number: P20963,2) or a CD3 zeta signaling domain as described in U.S. Patent No. 7,446,190.
[00268] [00268] For example, in some embodiments, the CAR includes a BCMA antibody or fragment, like any of the human BCMA antibodies, including sdAbs and scFvs, described herein, a spacer like any spacer containing lg joints, a transmembrane domain CD28, an intracellular signaling domain of CD28 and a signaling domain of CD3 zeta. In some embodiments, the CAR includes the BCMA antibody or fragment, like any of the human BCMA antibodies, including sdAbs and scFvs described herein, a spacer like any spacer that contains lg articulation, a CD28 transmembrane domain, a domain of 4-1BB intracellular signaling and a CD3 zeta signaling domain. In some embodiments, these CAR constructs further include a T2A ribosomal hop element and / or a tEGFR sequence, for example, downstream of the CAR.
[00269] [00269] In certain embodiments, multispecific binding molecules, for example, multispecific chimeric receptors, such as multispecific CARs, may contain any of the multispecific antibodies, including, for example, bispecific antibodies, multispecific single chain antibodies, for example for example, diabodies, triacodies and tetrabodies, tandem di-scFvs and tandem tri-scFvs, as described above in Section | .A.
[00270] [00270] In some respects, antibodies or their antigen-binding fragments, in the provided CARs, have one or more specified functional features, such as binding properties, including recognition or binding to specific epitopes, such as epitopes that are similar or overlap with those specifically | bound by other antibodies, such as reference antibodies, or epitopes other than those specifically bound by other antibodies, such as reference antibodies, the ability to compete for binding with other antibodies, such as reference antibodies and / or specific binding affinities. In other modalities, the antibodies or their antigen-binding fragments, in the provided CARs, recognize, as they specifically recognize, or bind, for example, specifically bind, to epitopes that are different or do not overlap with those specifically linked by other antibodies as reference antibodies. For example, epitopes specifically bound by antibodies, in the CARs provided, are different from those specifically bound by other antibodies, such as the reference antibodies. In some embodiments, antibodies and their antigen-binding fragments do not compete directly, or compete to a lesser extent, with binding to other antibodies, such as the reference antibodies.
[00271] [00271] In some embodiments, the antibodies or their antigen-binding fragments recognize or specifically bind to the BCMA protein. In any of the modalities, an antibody or antigen-binding fragment, in the provided CARs, which specifically recognize BCMA, specifically binds to BCMA. In some embodiments provided herein, the BCMA protein refers to human BCMA, a mouse BCMA protein or a non-human primate BCMA protein (for example, cymbal monkey). In some embodiments of any of the modalities here, the BCMA protein refers to the human BCMA protein. The observation that an antibody or other binding molecule binds to the BCMA protein or specifically binds to the BCMA protein does not necessarily mean that it binds to a BCMA protein of all species. For example, in some embodiments, BCMA protein-binding characteristics, such as the ability to specifically bind to it and / or compete for binding to a reference antibody and / or to bind to a specific affinity or compete in a a certain degree, in some modalities, refers to the capacity in relation to a human BCMA protein, and the antibody may not have this resource in relation to a BCMA protein of another species, such as mouse.
[00272] [00272] In some embodiments, the antibody or antigen binding fragment binds to a mammalian BCMA protein, including naturally occurring BCMA variants, such as certain binding variants or allelic variants.
[00273] [00273] In some embodiments, antibodies specifically bind to the human BCMA protein, such as an epitope or region of the human BCMA protein, such as the human BCMA protein comprising the amino acid sequence of SEQ ID NO: 367 ( Gen- Bank No.
[00274] [00274] In some embodiments, the binding antigen to the supplied antigen or CAR exhibits preferential binding to membrane bound BCMA as compared to soluble BCMA. In some modalities, the binding antigen to the supplied antigen or CAR exhibits higher binding affinity to membrane bound BCMA compared to soluble BCMA.
[00275] [00275] In one embodiment, the extent of binding of an anti- <BCMA antibody or antigen or CAR binding domain to an unrelated non-BCMA protein, such as a non-human BCMA protein or other non-BCMA protein, is less than or about 10% of antibody binding or antigen or CAR binding domain to human BCMA protein or human membrane bound BCMA measured, for example, by a radioimmunoassay (RIA). In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies or antigen-binding domains or CARs in which the binding to the mouse BCMA protein is less than or equal to or about 10% of the binding of the antibody to the human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the CARs provided, are the antibodies in which the binding to the BCMA protein of the cynomolgus monkey is less than or equal to or about 10% of the antibody binding to the human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the CARs provided, there are antibodies in which the binding to the BCM protein of monkey cynomolgus and / or a mouse BCMA protein is similar or about the same as antibody binding to human BCMA protein. In some embodiments, among the antibodies or antigen binding domains in the provided CARs, are antibodies or antigen binding domains or CARs in which the binding to the soluble BCMA protein is less than or equal to about 10% of the binding. antibody to the membrane-bound BCMA protein.
[00276] [00276] In some embodiments, the antibody specifically binds and / or competes to bind it with a reference antibody and / or binds to a specific affinity or competes to a certain degree with a protein of BCMA, for example, human BCMA, mouse BCMA protein or a non-human primate BCMA protein (for example, cynomolgus monkey).
[00277] [00277] In some embodiments, the antibodies, in the CARs provided, are able to bind to the BCMA protein, such as the human BCMA protein, with at least a certain affinity, as measured by any of several known methods. In some modalities, affinity is represented by a constant decoupling equilibrium (Ko); in some modalities, affinity is represented by ECs50o.
[00278] [00278] A variety of assays are known to assess binding affinity and / or determine whether a binding molecule (eg, an antibody or fragment) specifically binds to a specific ligand (eg, an antigen, as a BCMA protein). It is within the skill of someone skilled in the art to determine the binding affinity of a binding molecule, for example, an antibody, to an antigen, for example, BCMA, such as human BCMA or human BCMA or BCMA of cinomolgo or BCMA of mundane, as the use of any of a number of binding assays that are well known in the art. For example, in some modalities, a BIAcore & instrument can be used to determine the binding kinetics and constants of a complex between two proteins (for example, an antibody or fragment thereof and an antigen).
[00279] [00279] SPR measures changes in the concentration of molecules on the surface of the sensor, as the molecules attach or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thus allowing the measurement of the connection kinetics between two molecules. The dissociation constant for the complex can be determined by monitoring changes in the refractive index in relation to the time the buffer is passed over the chip. Other tests suitable for measuring the binding of one protein to another include, for example, immunoassays, such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding, monitoring the change in spectroscopic or optical properties of proteins through fluorescence, UV absorption, circular dichroism or nuclear magnetic resonance (NMR). Other specimen tests include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detecting expressed polynucleotides or protein binding.
[00280] [00280] In some embodiments, the binding molecule, for example, antibody or fragment thereof or the antigen-binding domain of a CAR, binds, as it specifically binds, to an antigen, for example, a protein BCMA or an epitope in it, with an affinity or KA (that is, an equilibrium association constant of a specific bonding interaction with 1 / M units; equal to the ratio of the on [Kon or kal rate, and the off rate [Kkor or ka] for this association reaction, assuming bimolecular interaction) equal to or greater than 105º M !. In some embodiments, the antibody or fragment of the same or antigen-binding domain of a CAR exhibits a binding affinity for the peptide epitope with a Kp (ie, an equilibrium dissociation constant for a specific binding interaction with units of M; equal to the ratio of the off rate [kor or ka] to the on rate [Kon Ou ka] for this association reaction, assuming bimolecular interaction) equal to or less than 10º M. For example, the dissociation constant of Kp balance ranges from 10º M to 103º M, as 107 M to 107º M, 108º M to 10º M or 10º M to 107º M. The on rate (constant of the association rate; Kon or ka; 1 / Ms units), and the off rate (constant of the dissociation rate; Korr or Ka; 1 / s units) can be determined using any of the test methods known in the art, for example, surface plasmon resonance (SPR).
[00281] [00281] In some embodiments, the binding affinity (ECso) and / or the antibody dissociation constant (eg, antigen-binding fragment) or antigen-binding domain of a CAR for the BCMA protein, such as human BCMA protein, is either about 0.01 nM to about 500 nM, or about 0.01 nM to about 400 nM, or about 0.01 nM to about 100 nM , from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or about from 0.01 nM to about 0.1 nM, is either about 0.1 nM to about 500 nM, or about 0.1 nM to about 400 nM, about or about 0 , 1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about from 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or about 1 nM to about 50 nM, of or from about 1 nM to about 10 nM, or from ut 2 nM to about 50
[00282] [00282] In some embodiments, the binding affinity can be classified as high affinity or low affinity. In some cases, the binding molecule (for example, antibody or its fragment) or antigen-binding domain of a CAR that exhibits low to moderate affinity binding displays a KA of up to 107 Y ', up to 106 M, up to 10th M1. In some cases, a binding molecule (antibody or fragment thereof) that exhibits high affinity binding to a specific epitope interacts with that epitope with a KA of at least 107 M ”, at least 108º M, at least 10º M !, at least 10 * º M ”, at least 10º! M, at least 10 ! M or at least 10 " M !. In some embodiments, the binding affinity (EC50o) and / or the equilibrium dissociation constant, Ko, of the binding molecule, for example, anti-BCMA antibody or its fragment or antigen-binding domain
[00283] [00283] In some embodiments, the binding affinity of a binding molecule, such as an anti-BCMA antibody or antigen-binding domain of a CAR, for different antigens, for example, BOCMA proteins of different species can be compared to determine the species' cross-reactivity. For example, species cross-reactivity can be classified as high cross-reactivity or low cross-reactivity. In some modalities, the equilibrium dissociation constant, Kp, for different antigens, for example, BCMA proteins from different species, such as human, cynomolgus monkey or mouse, can be compared to determine species cross-reactivity. In some modalities, the species cross-reactivity of an anti- <BCMA antibody or antigen-binding domain of a CAR may be high, for example, the anti-BCMA antibody binds to human BCMA and a variant of BCMA species to a similar degree, for example, the ratio of Kp to human BCMA and Kp to the species variant of
[00284] [00284] In some embodiments, the binding affinity of the anti-BCMA antibody or antigen binding domain of a CAR, for different form or topological type of antigens, for example, soluble BCMA protein compared to the affinity of binding to a membrane-bound BCMA to determine the preferred binding or relative affinity for a specific topological shape or type. For example, in some respects, the provided anti-BCMA antibodies or antigen-binding domains may exhibit preferential binding to membrane-bound BCMA over soluble BCMA and / or exhibit increased membrane-bound BCMA affinity with - comparison to soluble BCMA. In some embodiments, the equilibrium dissociation constant, Kp, for different forms or topological types of BCMA proteins, can be compared to determine preferential binding or relative binding affinity. In some embodiments, the preferential binding or affinity relative to a membrane bound BCMA compared to soluble BCMA may be high. For example, in some cases, the ratio of Ko to soluble BCMA, and the Kp to membrane bound BCMA is greater than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the antibody or antigen binding domain preferentially binds or has a greater binding affinity to membrane bound BCMA. In some cases, the ratio of Ka to membrane-bound BCMA and Ka to soluble BCMA is greater than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the antibody or antigen binding domain preferentially binds or has a greater binding affinity to membrane bound BCMA. In some cases, the antibody or CAR antigen-binding domain binds soluble BCMA and membrane-bound BCMA to a similar degree, for example, the ratio of Ko to soluble BCMA and Kp to BCMA | attached to the membrane is or is about 1. In some cases, the antibody or CAR antigen-binding domain binds to soluble BCMA and membrane-bound BCMA to a similar degree, for example, the ratio of Ka to soluble BCMA and Ka to Membrane-bound BCMA is or is about 1. The preferred degree of binding or relative affinity for membrane-bound BCMA or soluble BCMA can be compared to that of a known antibody, such as a reference antibody.
[00285] [00285] In some embodiments, the antibodies or their antigen-binding fragments, in the CARs provided, bind to a degree similar to a human BCMA protein and a non-human BCMA protein or other non-BCMA proteins. For example, in some embodiments, the antibodies or their antigen-binding fragments or the antigen-binding domain of a CAR bind to a human BCMA protein, such as the human BCMA protein that comprises the amino acid sequence of SEQ ID NO: 367 (GenBank No. BAB60895.1) or SEQ ID NO: 368 (NCBI NP 001183,2) or an allelic or bonding variant, with an equilibrium dissociation constant (Ko), and a non-human BCMA , as a BCMA of a cinomolgo monkey, as the protein of
[00286] [00286] In some embodiments, the antibodies or their antigen-binding fragments, in the CARs provided, bind to a degree similar to a soluble BCMA protein, and to a membrane-bound BCMA protein, with a constant equilibrium dissociation (Ko) that is similar or almost the same or less than 2 times difference or less than 5 times difference.
[00287] [00287] For example, in some embodiments, the antibodies, in the provided CARs or their antigen-binding fragments, bind to a human BCMA with a Kp of about or less than about 1 µM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 NM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 NM, 6 NM, 5 NM, 4 NM, 3 NM, 2 NM or 1 nM or less and binds to a cynomolgus monkey BCMA with a Kp of about or less than or about 1 uM, 500 nM, 100 nM, 50 nM, 40 nM, nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 NM, 12 nM, 11 nM, 10 nM, 9 nM, 8 NM, 7 NM, 6 NM, 5 NM, 4 NM, 3 AM, 2 nM or 1 nM or less. In some embodiments, the antibodies or their antigen-binding fragments bind to a mouse BCMA protein with a Kp of about or less than about 1 µM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM , 25 nM, 20 nM, 19 NM, 18 nM, 17 NM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 NM, 6 NM, 5 NM, 4 NM, 3 NM, 2 NM or 1 NM or less. In some modalities, the antibodies or their antigen-binding fragments, in the CARs provided, bind to a human BCMA, to a cynomolgus monkey BCMA, and to a high-affinity mouse BCMA. In some embodiments, antibodies or their antigen-binding fragments bind to a human BCMA and to the BCMA of a cynomolgus monkey with high affinity, and to a mouse BCMA with low affinity. In some embodiments, antibodies or their antigen-binding fragments bind to a human BCMA and BCMA from other species, or other variants of the BCMA protein, with high affinity.
[00288] [00288] In some embodiments, the total binding capacity (Rmax), measured using specific surface plasmon resonance (SPR) conditions, is used to determine the binding ability or capacity of the antibody or its binding fragment antigen to antigen, for example, a BCMA protein, such as a human BCMA protein. For SPR analysis, the "ligand" is the target molecule immobilized on the sensor surface, for example, a BCMA protein, and the "analyzed" is the tested molecule, for example, antibody, for binding to the "ligand" . For example, the "analyzed" can be any of the antibodies, or their antigen-binding fragments, which bind to a BCMA protein. For a ligand pair and analyzed in the SPR, the Rmax can be determined by assuming a 1: 1 connection stoichiometry model, for a specific condition. The binding capacity (Rmax) was determined using the following formula: Rmax (RU) = (molecular weight of the analyzed) / (molecular weight of the ligand) x level of immobilized ligand (RU). For example, under particular SPR conditions, the Rmax of binding between any antigen-binding antibody or fragment and a BCMA protein, such as a human BCMA or a cynomolgus BCMA, is at least or at least about 50 resonance units (RU), such as about 25 RU, 20 RU, 15 RU, 10 RU, 5 RU or 1 RU.
[00289] [00289] In some embodiments, antibodies, such as human antibodies, in the CAR provided, specifically bind to an epitope or specific region of the BCMA protein, as generally an epitope or extracellular region. The BCMA protein is a 184 amino acid protein with type III membrane that contains an extracellular domain, a transmembrane domain and a cytoplasmic domain. With reference to a human BCMA amino acid sequence mentioned in SEQ ID NO: 367, the extracellular domain corresponds to amino acids 1-54, amino acids 55-77 correspond to the transmembrane domain and amino acids 78-184 correspond to cytoplasmic domain.
[00290] [00290] Among the CARs provided are CARs that exhibit antigen-dependent activity or signaling, that is, signaling activity that is measurably absent or at baseline levels in the absence of antigen, for example, BCMA. Thus, in some respects, as long as CARs do not exhibit, or exhibit no more than a base or a tolerable or low level of tonic signaling or antigen independent activity or signaling in the absence of antigen, for example, BCMA, being present. In some embodiments, the anti-BCMA CAR expression cells provided exhibit biological activity or function, including cytotoxic activity, cytokine production and the ability to proliferate.
[00291] [00291] In some embodiments, the biological activity or functional activity of a chimeric receptor, such as cytotoxic activity, can be measured using any of several known methods. The activity can be evaluated or determined in vitro or in vivo. In some embodiments, the activity can be assessed once the cells are administered to the individual (for example, human). The parameters to be assessed include specific binding of a modified or natural T cell or another cell immune to the antigen, for example, in vivo, for example, by image or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the modified cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32 (7) : 689-702 (2009) and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004). In certain modalities, the biological activity of cells can also be measured by testing the expression and / or secretion of certain cytokines, such as interleukucin-2 (IL-2), interferon-gamma (IFNy), interleukin-4 (IL - 4), TNF-alpha (TNFa), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte and macrophage colony stimulating factor (GM -CSF), CD107a and / or TGF-beta (TGFB). Assays for measuring cytokines are well known in the art and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric beads matrix, RT-PCR, ELISPOT, flow cytometry and bioassays in which cells are responsive to the relevant cytokine are tested for responsiveness (eg proliferation) in the presence of a test sample. In some aspects, biological activity is measured by assessing the clinical outcome, such as a reduction in tumor overload or load.
[00292] [00292] In some respects, a reporter cell line can be employed to monitor antigen-independent activity and / or tonic signaling through anti-BCMA CAR expression cells. In some embodiments, a T cell line, such as a Jurkat cell line, contains a reporter molecule, such as a fluorescent protein or another detectable molecule, such as a red fluorescent protein, expressed under the control of the regulatory elements of the endogenous transcription Nur77. In some modalities, the expression of the reporter Nur77 is an intrinsic cell and dependent on signaling through a recombinant reporter containing a primary activation signal in a T cell, a signaling domain of a receptor component. T-cell (TCR) and / or signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), such as a CD37 chain. Nur77 expression is generally not affected by other signaling pathways, such as cytokine signaling or toll-like receptor (TLR) signaling, which can act in an extrinsic cellular manner and may not depend on signaling via the recombinant receptor. Thus, only cells that express the exogenous recombinant receptor, for example, the anti-BCMA CAR, containing the appropriate signaling regions, are capable of expressing Nur77 by stimulation (for example, specific antigen binding). In some cases, Nur77 expression may also show a dose-dependent response to the amount of stimulation (eg, antigen).
[00293] [00293] In some embodiments, the anti-BCMA CARs provided exhibit improved expression on the cell surface, as compared to an alternative CAR that has an identical amino acid sequence, but is encoded by an eliminated non-binding site and / or a codon-optimized nucleotide sequence. In some embodiments, the expression of the recombinant receptor on the cell surface can be evaluated. Methods for determining the expression of the recombinant receptor on the cell surface may include the use of antibodies specific for the chimeric antigen (CAR) receptor (eg, Brentjens et a /., Sci. Transl. Med. 2013 Mar; 5 ( 177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitopic markers and monoclonal antibodies that specifically bind to a CAR polypeptide (see, Ordering Pub. international patent No. WO2014190273). In some modalities, the expression of the recombinant receptor on the cell surface, for example, primary T cell, can be assessed, for example, by flow cytometry, using binding molecules that can bind to the recombinant receptor or a portion that can be detected
[00294] [00294] In certain embodiments, BCMA-binding molecules, for example, antibodies or polypeptides, such as chimeric receptors containing them, are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, for example, bispecific antibodies. Multispecific binding partners, for example, antibodies, have binding specificities for at least two different sites, which may be on the same or different antigens. In certain embodiments, one of the binding specificities is for BCMA, and the other is for another antigen. In some embodiments, additional binding molecules bind and / or recognize a third or more antigens. In certain embodiments, bispecific antibodies can bind to two different BCMA epitopes. Bispecific antibodies can also be used to locate cytotoxic agents in BCMA expression cells. Bispecific antibodies can be prepared as complete antibodies or antibody fragments. Multispecific antibodies include single chain multispecific antibodies, for example, diabody, tribody and tetribody, tandem di-scFvs and tandem tri-scFvs. Multispecific chimeric receptors, such as multispecific CARs, are also provided, containing antibodies (for example, antigen-binding fragments). Multispecific cells containing antibodies or polypeptides, including the same, are also provided as cells containing a cell surface protein including anti-BCMA antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope in BCMA.
[00295] [00295] “Examples of antigens include specific B cell antigens, other tumor specific antigens, such as antigens expressed specifically or associated with a leukemia (eg, B cell leukemia), lymphoma (eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.) or myeloma, for example, multiple myeloma (MM), a malignancy of plasma cells (for example, plasmacytoma). For example, antigens include those expressed specifically or associated with chronic B-cell lymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL) , Burkitt's lymphoma (for example, endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma), lining cell lymphoma (MCL), non-small cell lung cancer (NSCLC), chronic (or myelogenous) myeloid (CML) ), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), marginal zone lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), anaplastic large cell lymphoma (ALCL), lymphoma refractory follicular, Wal-denstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, lymphoma of the mucosa-associated lymphatic tissue (MALT), marginal zone lymphoma, nodal monocytoid B-cell lymphoma, immunoblastic lymphoma, lymphoma large cell, lymphoma di mixed cell spindle, angiocentric pulmonary B cell lymphoma, pulmonary lymphocytic lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, cancer breast cancer, squamous cell epithelial cancer, melanoma, myeloma as a
[00296] [00296] In some modalities, between the second or additional antigens for multiple targeting strategies includes those in which at least one of the antigens is a universal tumor antigen or a member of the same family. In some modalities, the second or additional antigen is an antigen expressed in a tumor. In some embodiments, the BCMA-binding molecules provided here target an antigen on the same type of tumor as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a type of tumor.
[00297] [00297] Second exemplary or additional antigens include CDA, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80 , CD126, CD138, B7, MUC-1, la, HM1,24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, fibronectin ED-B, an oncogene, an oncogene product, CD66a-d, antigens necrosis, li, IL -2, T101, TAC, IL-6, ROR1I, TRAIL-R1 (DR4), TRAIL-R2 (DR5), tEGFR, Her2, L1I-CAM, mesothelin, CEA, hepatitis surface antigen B, antifolate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa 2, ErbB2, ErbB3, ErbB4, erbB dimers, EGFR villl, FBP, FCRLS5,
[00298] [00298] In some respects, the antigen, for example, the second or additional antigen, such as the disease-specific antigen and / or the related antigen, is expressed in multiple myeloma, as the D member of group 5 of the coupled receptor to G class C protein (GPRC5D), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24), BAFF -R, TACI and / or FcRH5. Other anti-
[00299] [00299] “Methods are provided here to optimize polynucleotides for expression and / or therapeutic use, and polynucleotides optimized, for example, according to the methods. In some embodiments, the methods or optimizations provided reduce the heterogeneity and / or increase the homogeneity of the transcribed RNA, such as messenger RNA (mMRNA), for example, when the polynucleotide is expressed in a cell, as in a cell type specific, as in a mammal, for example, human cell type, such as a human T cell, such as a primary human T cell or T cell lineage
[00300] [00300] Genomic nucleic acid sequences generally, in nature, in a mammalian cell, undergo co-transcriptional processing or immediately after transcription, in which a nascent precursor of messenger ribonucleic acid (pre-mRNA), transcribed from a genomic sequence of deoxyribonucleic acid (DNA), is in some cases edited by means of ligation, to remove the introns, followed by the exons binding in eukaryotic cells. Consensus sequences for binding sites are known, but in some respects, specific nucleotide information that defines a binding site can be complex and may not be easily apparent based on the methods available. Cryptic binding sites are binding sites that are not predicted based on standard consensus sequences and are activated in varying ways. Therefore, the variable processing of pre-mRNA at the cryptic binding sites leads to heterogeneity in the mRNA products transcribed after expression in eukaryotic cells.
[00301] [00301] Polynucleotides generated for the expression of transgenes are typically constructed from sequences of nucleic acids, such as complementary DNA (cDNA), or portions thereof, which do not contain introns. Thus, it is not expected that the joining of such sequences will occur. However, the presence of cryptic binding sites within the cDNA sequence can lead to unintended or unwanted binding reactions and heterogeneity in the transcribed mMRNA. This heterogeneity results in the translation of unintended protein products, such as protein products truncated with variable sequences of amino acids that exhibit modified expression and / or activity.
[00302] [00302] “Methods and methods are also provided to determine the heterogeneity of a transcribed nucleic acid, such as one that encodes or contains a transgene or that encodes a recombinant protein. In some embodiments, the methods include determining the heterogeneity of a transcribed nucleic acid sequence that includes all or a portion of the 5 '(5' RTU) untranslated region and / or all or 3 '(3') untranslated region RTU), of the transcribed nucleic acid. Also provided herein are methods for identifying the presence of binding sites, such as cryptic binding sites, based on the heterogeneity of the transcribed nucleic acid. Also provided are methods of identifying a transgene candidate for removal of binding sites, such as cryptic binding sites, using the methods provided to determine the transgene nucleic acid heterogeneity of the transgene. Methods are also provided to reduce the heterogeneity of an expressed transgene transcription.
[00303] [00303] “Also provided here are methods of identifying a recombinant transgene or protein or nucleic acid candidate for the removal or modification of one or more binding sites, such as cryptic binding sites, such as based on the determined heterogeneity of the acid transcribed nucleic acid, for example, from the transgene.
[00304] [00304] “Methods and methods are also provided to reduce the heterogeneity of a transcribed nucleic acid (for example, trans-
[00305] [00305] In some embodiments, the elimination of binding sites, such as cryptic binding sites, can improve or optimize the expression of a transgene product, such as a transgene-translated polypeptide, such as an anti-BCMA CAR polypeptide . Binding at cryptic binding sites of an encoded transgene, such as an encoded BMCA CAR molecule, can lead to reduced protein expression, for example, cell surface expression and / or reduced function, for example, intracellular signaling reduced. Polynucleotides encoding anti-BMCA CAR proteins that have been optimized to reduce or eliminate cryptic binding sites are provided here. Polynucleotides are also provided here that encode anti-BCMA CAR proteins that have been optimized for codon expression and / or in which one or more sequences, such as one identified by the methods or is present and / or in which a site identified link, like any of the link sites identified here, is not present. Among the polynucleotides provided are those that exhibit below a certain degree of RNA heterogeneity or binding forms when expressed under certain conditions and / or introduced into a specified cell type, such as a human T cell, such as a human T cell primary, and cells and compositions and articles of manufacture containing these polypeptides and / or
[00306] [00306] In some embodiments, reducing the RNA heterogeneity or removing the potential binding site comprises modifying a polynucleotide. In some embodiments, the modification includes one or more nucleotide modifications, such as a substitution or replacement, as compared to a reference polynucleotide, such as an unmodified polynucleotide that encodes the same polypeptide. In some embodiments, the reference polynucleotide is one in which the transcribed RNA (for example, mMRNA), when expressed in a cell, exhibits more or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more of RNA heterogeneity. In some embodiments, the methods provided may result in polynucleotides in which the RNA heterogeneity of the transcribed RNA is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50 % or more. In some embodiments, the methods provided produce polynucleotides in which the homogeneity of the transcribed RNA is at least 70%, 75%, 80%, 85%, 90% or 95% or greater. A. Methods of Measuring and Reducing RNA Heterogeneity
[00307] [00307] Methods, approaches and strategies for measuring, evaluating and / or reducing the heterogeneity of the RNA of a nucleic acid, such as a transcribed RNA, for example, when expressed in a specific cell type or context, are provided here, as well as polynucleotides that exhibit a reduction in such heterogeneity and / or risk, compared to a reference polynucleotide. In some modalities, a reference polynucleotide can be evaluated for RNA heterogeneity, as by methods described in this Section. In some embodiments, the methods provided involve identifying RNA heterogeneity (for example, mMRNA) or its probability, as in a specific cell or context, as due to cryptic binding sites.
[00308] [00308] RNA heterogeneity can be determined by any one of several methods provided here or described or known. In some embodiments, the RNA heterogeneity of a transcribed nucleic acid is determined by the amplification of the transcribed nucleic acid, such as by polymerase chain reaction with reverse transcriptase (RT-PCR) followed by the detection of one or more differences, such as size differences, in the single or more amplified products. In some modalities, the RNA heterogeneity is determined based on the number of amplified products of different sizes or the list of several amplified products of different sizes. For example, in some modalities, the RNA heterogeneity is quantified by determining the number, quantity, or ratio of amplified product of different size compared to the number or quantity of total amplified products. In some cases, all or substantially all of a specific transcript is determined to be of equal size, and in this case, the RNA heterogeneity is low. In some cases, a variety of transcripts of different sizes are present, or a large ratio of a specific transcript is of a different size compared to the predicted size of the amplified product without cryptic or unwanted binding events. In some embodiments, the RNA heterogeneity can be calculated by dividing the total number or quantity of all amplified products that are different in size compared to the expected size of the amplified product by the total number or quantity of all amplified products . In some modalities, the predicted size of the transcribed or amplified product is that of an RNA that does not contain or is not expected to contain a cryptic binding site. In some embodiments, the predicted size of the transcribed or amplified product takes into account one or more binding sites that are desired or intentionally placed.
[00309] [00309] In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is harvested from cells, expressing the transgene to be optimized and amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) using a primer specific for the 5 '(5' RTU) untranslated region, in some cases corresponding to a portion of the promoter sequence in the expression vector, located upstream of the transgene in the transcribed RNA and a specific primer for the 3 'untranslated region (3 'UTR), located downstream of the transgene expressed in the transcribed RNA sequence or a specific primer for a sequence within the transgene. In particular embodiments, at least one primer complementary to a sequence in the 5 'untranslated region (RTU) and at least one primer complementary to a sequence in the 3' untranslated region (RTU) are employed to amplify the transgene. An exemplary representation of the amplification of a transcript and resulting product using a forward primer specific for the 5 'RTU and a specific primer for a nucleotide sequence at the 3' RTU and a predicted amplified product, where no binding events occurred, at FIG. 21A. An exemplary representation of several exemplary amplified products (i.e., heterogeneity) resulting from the amplification of a transcription that has a 5 'RTU, with a transcribed promoter sequence that contains a known binding donor site (P-SD) and a site known binding receptor (P-SD), a transcribed transgene containing an unknown binding (cryptic) donor site (T-SD) and two unknown binding (cryptic) binding sites (T-SA) and one 3 'RTU, using specific primers for regions of the 5' RTU and 3 'RTU, is shown in FIG. 21B.
[00310] [00310] Exemplary primers specific for the 5 'untranslated region (RTU) include primers directed to sequences within the transgene promoter. In some examples, a specific primer for an EF1a / HTLV promoter. An exemplary front primer, specific for an EF1a-HTLV promoter is mentioned in SEQ ID NO: 763.
[00311] [00311] Examples of specific primers for the 3 'untranslated region (RTU) include primers targeting 3' post-transcriptional regulatory elements located downstream of the transgene. Examples of 3 'post-transcriptional regulatory elements include the groundhog hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), mentioned in SEQ ID NO: 636. An exemplary front starter, specific for a WPRE is mentioned in SEQ ID NO: 764.
[00312] [00312] In some modalities, several pairs of primers can be used to amplify the transgene, as for long transgenes. In some embodiments, sequential or nested pairs of forward and reverse primers, to fold a sliding window of amplified products, can be used to obtain complete and overlapping coverage of the sequence. Typically, primers are modified to amplify a transgene length that is about 1.5-6 kb, 2-6 kb or 3-6 kb. An exemplary representation of amplification of a transcript using nested primer pairs is provided in FIG. 21C.
[00313] [00313] The amplified nucleic acid sequence is then analyzed for heterogeneity in terms of amplified transcription lengths. In some examples, heterogeneity is determined by the number and intensity of the bands for the expressed sequence. In some modalities, RNA sequences having binding events after expression generate several bands with different mobility. In some modalities, a main band is detected in the expected mobility for a sequence that does not have any unpredictable binding events and 1 or more additional bands of varying intensity and mobility indicate the occurrence of one or more cryptic binding events within the sequence of transgene.
[00314] [00314] The technician versed in the technique can solve the RNA, like the messenger RNA, and analyze its heterogeneity by various methods. Exemplary non-limiting methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field flow fractionation and chromatography, such as size exclusion chromatography or liquid chromatography.
[00315] [00315] One or more steps of the above techniques can be performed under denaturing conditions, partially denaturating conditions or non-denaturing conditions. Denaturation conditions can include conditions that cause denaturation of nucleic acid transcription (eg, mRNA) due to temperature, chaotropic agents (including salts), organic agents, among other denaturation mechanisms. With thermal denaturation conditions, a high temperature can be applied. The elevated temperature may be sufficient to denature the intramolecular hydrogen bonds, cause a change or loss of the secondary or tertiary structure, and so on. For example, conditions of temperature or thermal denaturation may include a temperature of 25 degrees Celsius to 95 degrees Celsius, 35 to 85 degrees Celsius, 55 to 75 degrees Celsius or another range within those ranges. Likewise, higher or lower temperatures can be used as appropriate to cause the desired level of denaturation. Conditions of temperature or thermal denaturation may also be dependent on the identity of the nucleic acid transcription, so that different temperatures are used for different nucleic acid transcripts or types of nucleic acid transcripts. Denaturation conditions may also include the use of chaotropic agents, such as lithium perchlorate and other perchlorate salts, guanidinium chloride and other guanidinium salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea or others. Denaturation conditions can also include organic denaturing agents, such as dimethylsulfoxide (DMSO), acetonitrile and glyoxal. In addition, denaturation conditions may include a combination of two or more of these types of denaturation conditions. Any one or more of the steps in the RNA heterogeneity determination techniques can be performed at an elevated temperature or at room temperature, with or without organic or chaotropic agents.
[00316] [00316] In some modalities, the RNA transcription topology, and the apparent size (hydrodynamic) can be analyzed by gel electrophoresis, such as agarose gel electrophoresis. In some examples, RNA transcription can be resolved on a 0.05% to 2% agarose gel, such as a 1.2% agarose gel, and visualized by staining or using specific probes for a specific sequence. In some modalities, RNA transcripts can be evaluated directly by gel electrophoresis, or they can be evaluated after amplification, as quantitative amplification methods. Nucleic acid stains for visualizing nucleic acid on the agarose gel are well known. Exemplary stains include the BlueView "" nucleic acid stain (Millipore Sigma), SYBRO Gold nucleic acid stain (ThermoFisher), SYBRO green nucleic acid stain (Millipore Sigma), SYBRO Green | (ThermoFisher), PicoGreen & nucleic acid stain (Invitrogen) and ethidium bromide: 0.5 ug / mL, prepared in distilled water or embedded in the gel. In some examples, the nucleic acid is stained using the Quant-4iT "y" PicoGreenO bond, followed by fluorescence detection and quantification of the amplified products. The agarose gel method provides a more quantitative, but less resolutive, measure of size distribution. In some embodiments, nucleic acid fragments, resolved by agarose gel electrophoresis, can be visualized by Northern blot for RNA or Southern blot for amplified reverse transcriptase-polymerase chain reaction (RT-PCR) products.
[00317] [00317] Chip-based capillary electrophoresis (for example, with AGILENT 2100 BIOANALYZER'Y) can be used as a quick and routine method to monitor the integrity of RNA transcripts and their distribution by size. The separation is based on size and hydrodynamic charge and is affected by the length of the nucleotides and the folded structure of RNA transcription. In one embodiment, the method includes releasing the sample into a chip channel with an electrolytic medium and applying an electric field to the chip that causes RNA transcription and impurities migrate through the channel. RNA transcription has an electrophoretic mobility different from impurities, so that RNA transcription migrates through the channel at a rate different from the rate at which impurities migrate through the channel. The electrophoretic mobility of RNA transcription is proportional to an ionic charge of RNA transcription and inversely proportional to the frictional forces in the electrolytic medium. The method also includes collecting from the chip, the sample comprising RNA transcription and one or more separate portions of the sample comprising the impurities. In addition, the method includes characterizing an aspect of at least a portion of the sample comprising RNA transcription and one or more separate portions of the sample comprising impurities. Characterization can include, for example, quantification of load variants. c) Analytical Ultracentrifugation (AUC)
[00318] [00318] Analytical ultracentrifugation (AUC) is a solution phase method to measure the molecular weight distribution, without the potential artifacts that could be introduced by the matrix interaction (resin or gel) in the SEC, agarose, or others methods. Both equilibrium AUC and sedimentation ultracentrifugation are used, and the latter provides sedimentation coefficients related to the size and shape of RNA transcription. An analytical BECKMAN'Y ultracentrifuge equipped with a UVW / visible scanning optics is used for analysis of RNA transcription. d) Field Flow Fractionation (FFF)
[00319] [00319] Another solution phase method to assess hydrodynamic size distribution is field flow fractionation (FFF). FFF is a separation technique in which a field is applied to a suspension or fluid solution pumped through a long, narrow channel, perpendicular to the flow direction, to cause the separation of the polynucleotides (RNA transcripts) present in the fluid, under the force exerted by the field. The field can be asymmetric flow through a semipermeable, gravitational, centrifugal membrane, thermal, electrical, magnetic gradient, etc. e) Chromatography
[00320] [00320] Chromatography can also be used to detect the heterogeneity of RNA transcription lengths. The methods of size exclusion chromatography and liquid chromatography to determine the heterogeneity of mMRNA are described in WO 2014/20144711, which is incorporated herein by reference. B. Polynucleotide Optimization Methods, for example, Po-
[00321] [00321] In some embodiments, the methods provided include optimizing and / or modifying the polynucleotide, for example, to reduce RNA heterogeneity and / or to remove or eliminate cryptic or unwanted binding sites. In some respects, methods are provided to reduce the heterogeneity of an express transgene transcription that involves the identification of a transgene candidate for the removal of binding sites, as by the methods described above in Section IA; identifying one or more potential binding donor and / or binding receptor sites; and modifying the nucleic acid sequence at or near one or more identified donor binding sites that have been identified, thereby generating a modified polynucleotide. In some ways, the methods also involve evaluating the transgenic candidacy for the removal of binding sites. In some embodiments, the methods also include repeating one or more steps above until the heterogeneity of the transcription is reduced compared to the initial heterogeneity of the transcription, as determined (as before the modification).
[00322] [00322] In some modalities, methods of reducing heterogeneity, such as removal or elimination of predicted binding sites, can be performed after codon optimization or in non-codon optimized RNA. In some respects, the methods involve identifying binding sites, such as one or more possible donors and / or binding sites, and modifying or altering the RNA sequence (for example, replacing or replacing one or more nuclei) at or near the binding site In some modalities, codon optimization can be performed before and / or after methods of reducing the heterogeneity of the transcribed RNA (for example, mMRNA), such as by removing or eliminating the binding sites In some embodiments, whether a transcript is a candidate for reducing RNA heterogeneity is determined based on the RNA heterogeneity measurement method, for example, as described in Section IL.A here. a transcribed nucleic acid that is detected as having heterogeneity is identified as a candidate for transgene to remove one or more binding sites. In some embodiments, a transgene sequence may be a candidate to reduce the heterogeneity when the transgene candidate's transcribed nucleic acid exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60 %, 65%, 70%, 75% or more heterogeneity after expression in a cell. In some modalities, after the transcription and processing of the polylucleotide in a human cell, optionally a human T cell, the messenger RNA (mMRNA) of the polynucleotide, exhibits at least 70%, 75%, 80%, 85 %, 90%, or 95% RNA homogeneity.
[00323] [00323] Methods are provided to reduce the heterogeneity of an expressed transgene transcription. In some embodiments, the methods involve identifying one or more potential binding donation sites and / or binding receptors, and modifying the nucleic acid sequence at or near one or more of the identified binding donation sites . In some modalities, the methods also involve the evaluation of the transgenic candidacy for removal of binding sites. In some respects, one or more steps described here can be repeated, for example, until the potential heterogeneity of the RNA is reduced compared to the initial or unmodified transcription. a) Identification of the Connection Site
[00324] [00324] In some aspects, the presence of possible cryptic binding sites (donor and / or receptor binding sites that are present in a transcription, such as a transgene transcription, may result in the heterogeneity of the transcription RNA after expression in a cell. In some embodiments, the methods involve identifying one or more potential binding sites that may be present in the transgene transcription, which are unwanted and / or that can be created in a transgene transcription from of several underlying sequences following codon optimization of a transcription and / or by mutation or mistake or transcription error In some aspects of the modalities provided, the donor binding sites and the binding receptor sites are identified independently. modalities, the recipient site (s) and / or link donor (s) is / are canonical (s), non-canonical site (s) and / or recipient (s) and / or cryptic donor (s).
[00325] [00325] In some embodiments, the methods provided include the identification of one or more potential binding sites (for example, canonical (s), non-canonical (s) and / or recipient (s) and / or donor site (s) cryptic (s) or branching sites) in a polynucleotide, such as a polynucleotide encoding a transgene, such as a recombinant receptor, which may exhibit RNA heterogeneity or contain unwanted ones. Polypeptides are also provided which have reduced numbers of such binding sites, compared to these reference polynucleotides.
[00326] [00326] In some ways, the identification of one or more binding sites in a nucleic acid sequence is an iterative process. In some embodiments, the binding sites can be identified using a binding site and / or codon optimization prediction tool, such as sending the initial or reference sequence encoding the transgene, as a receptor for binding to the transgene. BCMA, for example, anti-BCMA CAR, for a database, a gene synthesis provider or other source capable of comparing computationally or algorithmically the initial or reference sequence to identify or predict binding sites and / or to codon optimization and / or removal of the binding site. In some embodiments, after modifying the sequence for codon optimization and / or removal of the binding site, one or more additional evaluations of a sequence, such as a revised or modified nucleic acid sequence, are performed to further evaluate removing the binding site, such as cryptic binding sites, using one or more additional tools for predicting binding sites.
[00327] [00327] In some respects, the RNA heterogeneity may be the result of the spliceosome activity present in a eukaryotic cell. In some respects, ligation is typically performed in a series of reactions catalyzed by the spliceosome. Consensus sequences for binding sites are known, but in some respects, specific nucleotide information that defines a binding site can be complex and may not be readily apparent based on available methods. Cryptic binding sites are binding sites that are not predicted based on standard consensus sequences and are activated in a variable way. Therefore, the variable processing of pre-mRNA at the cryptic binding sites leads to heterogeneity in the transcribed mRNA products after expression in eukaryotic cells. In some cases, within the spliceosomal introns, a donor site (usually at the 5 'end of the intron), a branching site (near the 3' end of the intron) and a receptor site (3 'end of the intron) are needed for a link event. The donor binding site may include a GU sequence at the 5 'end of the intron, with a large, less highly conserved region. The binding receptor site at the 3 'end of the intron can terminate with an AG sequence.
[00328] [00328] In some embodiments, binding sites, including potential cryptic binding sites, can be identified
[00329] [00329] In some modalities, one or more prediction tools for link sites are selected for use in determining potential donors and / or link recipients. In some modalities, the tools for forecasting link sites that can be executed locally, which can be retrained with a set of data on the user's site; which can use databases for specific species (such as humans), which can be compiled
[00330] [00330] In some ways, binding site prediction tools can be used to identify a list of possible binding donors and / or binding receptor sites in a sequence, such as a polynucleotide sequence containing sequences of transgene. In some respects, the prediction tools can also generate one or more prediction scores for one or more sequences in the polynucleotide, which may indicate the probabilities of one or more sequences being a donor or receptor site sequence.
[00331] [00331] In some embodiments, the method involves comparing the prediction score for a specific binding site with a threshold score or reference score to determine or identify a particular binding site that is a candidate for elimination or removal. For example, in some embodiments, the predicted link site is identified as a potential link site when the forecast score is higher or lower than the threshold score or the reference score. In some respects, considerations for eliminating or removing a specific binding site include the prediction score compared to a reference score or a threshold score; and whether a specific binding site is desired or intentional (for example, when the binding event is most advantageous or is necessary for the regulation of transcription and / or translation). In some respects, the likelihood that the resulting link variant will lose the desired function or have the compromised function
[00332] [00332] In some embodiments, the methods provided involve the elimination or elimination of one or more binding donor site (s) and / or binding receptor (s), such as potential donor and / or recipient sites that may be involved in a cryptic binding event that is not desired or that results in undesirable RNA heterogeneity. In some embodiments, the elimination of one or more binding sites comprises modifying one or more nucleotides (for example, by substitution or replacement) at, containing or close to the donor and / or receptor binding sites that are candidates for removal. In some respects, a specific nucleotide within a codon that is at, contains, or is close to the binding site is modified (for example, replaced or replaced).
[00333] [00333] In some embodiments, the codon at or near the attachment site for modification comprises one or more codons that involve one or both of the two nucleotides at the potential attachment site (in some cases, referred to as the "attachment site codon" Link"). When potential binding is expected to occur between two nucleotides in a codon, the codon is the only codon from the binding site to that binding site. If the potential bond is expected to occur between two adjacent codons, for example, between the last nucleotide of the first codon, and the first nucleotide of the next codon, the two codons are codons at the binding site. For example, for binding sites that are expected to be at the boundaries of two codons, the two adjacent codes may be candidates for nucleotide modification. In some embodiments, the one or more codons comprise a codon at the connection site. In some embodiments, the one or more codons comprise the two codons at the binding site. In some embodiments, the method involves eliminating the potential binding donor site by modifying one or both codons of the binding site. In some embodiments, the method involves eliminating a potential binding donor / recipient site by modifying one or both of the binding site codes. In some embodiments, the one or both codons at the link site are not modified, for example, when there is no synonymous codon for the codon at the link site. In some embodiments, if synonymous codons are not available for the codon of the specific binding site, one or more nucleotides in a nearby codon can be modified. In some embodiments, one or more codons that are modified include a codon at the binding site, wherein the modification comprises changing one or both nucleotides at the binding site to a different nucleotide or different nucleotides. In some modalities, in some modalities, the method involves eliminating the donor binding site by modifying one or both codons of the binding site, where the modification does not change one or two of the nucleotides of the binding site to a different nucleotide, but a nearby nucleotide, for example, a part of a codon adjacent to the binding site, is modified. In some embodiments, nearby or adjacent nucleotides that can be modified include modifying a nucleotide that is part of a nearby or adjacent codon, such as a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the codon of the connection site.
[00334] [00334] In some cases, manual modification of polynucleotides can be employed, preserving the encoded amino acid sequence, to reduce the likelihood of a predicted binding site. In some embodiments, one or more of the predicted binding sites with at least 80%, 85%, 90% or 95% probability of a binding site are modified manually to reduce the likelihood of the binding event. In some embodiments, one or more changes are / are by nucleotide substitution or substitution of 1, 2, 3.4, 5.6 or 7 nucleotides. In some embodiments, the modification (s) is / are at the junction of the binding donor site or are at the junction of the binding receptor site. In some embodiments, at least one of the one or more nucleotide modifications is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues from the junction of the binding site of the receptor binding site and / or link donor. In some modalities, libraries of modified nucleic acid sequences may be less likely to be generated from cryptic binding sites. In some modalities, the link donor sites and the sites
[00335] [00335] In some embodiments, the method involves the elimination of one or more potential donor binding sites, modifying one or two codons of the binding site or one or more codons close or adjacent (for example, if a synonymous codon is not available for the codon of the connection site). In some embodiments, the method involves eliminating one or more potential receptor binding sites by modifying one or two codons from the binding site or one or more codons close or adjacent (for example, if a synonymous codon is not available) for the codon of the connection site). In some embodiments, the near or adjacent codon that is subject to modification includes a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the codon of the binding site, such as a codon that is within one, two, or three codons of the binding site. In some embodiments, the methods may include removing or eliminating a potential branching site for attachment. In some aspects, a nucleotide within the codon at or near the branching site can be modified, for example, replaced or replaced, to eliminate the cryptic bond and / or reduce the RNA heterogeneity. In some embodiments, the modification of one or more nucleotides may involve a substitution or replacement of one of the nucleotides that may be involved in the link (such as at the linker donor site, at the linker receptor site or at the branch site) , so that the amino acid encoded by the codon is preserved, and the substitution or replacement of nucleotides does not alter the polypeptide sequence that is encoded by the polynucleotide. In some cases, the third position on the codon is more degenerate than the other two positions. Thus, several synonymous codes can encode a particular amino acid (see, for example, Section II.B.2 below). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the cell species, into which the polynucleotide is introduced (for example, human). In some ways, the species is human. In some modalities, the one or more codons are replaced by a corresponding synonymous codon than those most used in species or synonymous codons that have a similar frequency of use (for example, the closest frequency of use) to the corresponding codon ( see, for example, Section | IB2 below).
[00336] [00336] In some modalities, the methods also involve the evaluation of the transgenic candidacy for the removal of binding sites, after the initial proposed modification. In some respects, the proposed modification can be evaluated again, to assess the proposed modification and to identify any other potential binding sites after codon modification and / or optimization. In some respects, after modifying the sequence for codon optimization and / or removal of the binding site, one or more additional evaluations of a sequence, such as a revised or modified nucleic acid sequence, are performed to better assess the removal of the binding site, as cryptic binding sites, using the same or one or more others or additional link site prediction tool (s). In some respects, the proposed modifications are considered for subsequent steps, and iterative optimization can be used. In some ways, the methods also include repeating any identification and / or modification step, for example, until the heterogeneity of the transcription is reduced compared to the heterogeneity of the transcription, as initially determined. In some modalities, an additional or different modification, such as with a different nucleotide substitution in the same codon or a modification in a different position or codon, can be made after an interactive estimation and evaluation. In some modalities, the corresponding different synonym codon can be used, such as the second most frequently used in the specific species or a codon that has a similar frequency of use (for example, the next closest frequency of use) than the corresponding codon (see, for example, Section II.B.2 below).
[00337] [00337] In some respects, a proposed modification can be further evaluated, for example, to assess whether the modification generates an unwanted or additional restriction site in the polynucleotide. In some respects, an additional restriction site may not be desired, and an additional or different modification (for example, with a different nucleotide substitution on the same codon or a modification on a different position or codon) can be considered . In some respects, a specific restriction site, such as a designated restriction site, is avoided. In some respects, if the modification does not substantially reduce or, the forecast score of the link site, an additional or alternative modification may be proposed. In some modalities, the forecast score of the link site may be reduced or decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70% or 75%, after one or more iterations of the methods.
[00338] [00338] In some modalities of any of the methods provided here, a computer system can be used to execute one or more steps, tools, functions, processes or scripts.
[00339] [00339] Exemplary steps, functions, processes or scripts of the methods provided to identify and / or remove possible connection sites include one or more steps of: sequence selection, recording sequences in FASTA format, loading the codons (for example, from www.Kazusa.or.jp/codon, running GenesSplicer, loading forecasts, analyzing codons, determining overlaps in the forecast, identifying the next codon synonymous with greater use, reviewing the restriction site, creating annotations or evaluating other codons Specific steps can evaluate the direct and reverse chains In some respects, modifications to the link site noted earlier can also be considered, to allow for iterative optimization. In some modalities, any one or more of steps, functions, processes or scripts can be repeated.
[00340] [00340] In certain modalities, the methods provided here can be practiced, at least in part, with computer system configurations, including single processor or multiprocessor computer systems, minicomputers, mainframe computers, in addition to personal computers, devices portable computing, consumer based and / or programmable electronic microprocessors and the like, each of which can communicate operationally with one or more associated devices. In particular modes, the methods provided here can be practiced, at least in part, in distributed computing environments, so that certain tasks are performed by remote processing devices that are connected through a communications network. . In a distributed computing environment, program modules can be located on local and / or remote memory storage devices. In particular modalities, some or all of the steps of the methods provided here, can be practiced on independent computers.
[00341] [00341] In particular modalities, some or all of the steps of the methods provided here may operate in the general context of instructions executable by computer, such as program modules, plugs and / or scripts executed by one or more components. Generally, program modules include routines, programs, objects, data structures and / or scripts, which perform specific tasks or implement specific abstract data types. Normally, the functionality of the program modules can be combined or distributed as desired. In certain embodiments, instructions operable to make the processor perform one or more steps of the methods provided herein can be incorporated into a computer-readable medium with computer-executable instructions and transmitted as signals manufactured to transmit such instructions, as well as such as the results of executing instructions, for example, on a network. In some modalities, computer systems, computer-readable instructions, software, systems, networks and / or devices for executing or executing one or more steps of the methods provided here are also provided.
[00342] [00342] In some modalities, polynucleotides are modified by optimizing the codons for expression in humans. In some aspects, codon optimization can be considered before and / or after the steps for identifying the binding site and / or eliminating the binding site and / or in each of the iterative steps to reduce the RNA heterogeneity. Codon optimization generally involves balancing the percentages of selected codons with the abundance, for example, published abundance, of human transfer RNAs, for example, so that none is overloaded or limiting. In some cases, this balance is necessary or useful because most amino acids are coded for by more than one codon, and the use of codons generally varies from organism to organism. Differences in the use of codons between transfected or transduced genes or nucleic acids and host cells can have effects on the protein expression of the nucleic acid molecule. Table 3 below presents a table of frequency of use of exemplary human codons. In some embodiments, to generate codon-optimized nucleic acid sequences, the codons are chosen to select the codons that are in balance with the frequency of human use. The codon redundancy for amino acids is such that different codes code for an amino acid, as shown in Table
[00343] [00343] For example, the codons TCT, TCC, TCA, TOG, AGT and AGC code for Serine (note that T in DNA is equivalent to U in RNA). From a frequency of use of human codons, as shown in Table 3 above, the corresponding frequencies of use for these codons are 15.2, 17.7, 12.2, 4.4, 12.1 and 19.5 , respectively. As the TCG corresponds to 4.4%, if this codon were commonly used in the synthesis of genes, the tRNA for this codon would be limiting. In codon optimization, the objective is to balance the use of each codon with the normal frequency of use in the species of animals in which the transgene is intended to be expressed. C. Optimized Anti-BCMA CAR
[00344] [00344] In some embodiments, an initial or reference sequence encoding a transgene, such as a BCMA-binding receptor, for example, anti-BCMA CAR, is evaluated for codon optimization and / or removal of the binding site .
[00345] [00345] In some embodiments, the methods are performed on an anti-BCMA CAR, such as a CAR that contains a binding domain
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[00346] [00346] In some embodiments, the resulting modified nucleic acid sequence (s) is / are then synthesized and used to transduce the cells to test for binding as indicated by the heterogeneity of the RNA. Exemplary methods are as follows and described in the Examples. Briefly, RNA is harvested from the expressing cells, amplified by polymerase chain reaction with reverse transcriptase (RT-PCR) and resolved by agarose gel electrophoresis to determine the RNA heterogeneity, compared to the initial sequence. In some cases, improved sequences may be forwarded to the gene synthesis provider for further codon optimization and removal of the binding site, followed by further evaluation, modification, synthesis and testing of the cryptic binding site, until the RNA in the agarose gel exhibits minimal RNA heterogeneity.
[00347] [00347] In some embodiments, the methods provided to optimize a coding nucleic acid sequence encoding a transgene, such as an anti-BCMA CAR provided here or a construct provided here, are to reduce or eliminate the sites of cryptic bonding (see, for example, SEQ ID NO: 622 for an optimized codon spacing sequence optimized and eliminated at the binding site) and optimizing the use of human codon (see, for example, SEQ ID NO: 855 for an optimized spacer sequence and example spacer). An exemplary optimization strategy is described in the examples.
[00348] [00348] In some embodiments, polynucleotides are provided that encode a chimeric antigen receptor, comprising the nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes BCMA, including any of the binding domains the antigen described below; (b) a spacer of at least 125 amino acids in length; (c) a donor
[00349] [00349] In some embodiments, polynucleotides are provided that encode a chimeric antigen receptor, comprising the nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes BCMA, including any of the binding domains the antigen described below; (b) a spacer, wherein the encoding nucleic acid is or comprises, or consists or consists essentially of the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments, the antigen-binding domain comprises a Vx region and a Vi region that comprise the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence with at least 90% identity with SEQ ID NOS: 617 and 618, respectively.
[00350] [00350] Examples of modified polynucleotides, including polynucleotides that have been modified for codon optimization (O) and / or binding site elimination (SSE), are also provided here. Examples of such polynucleotides are shown in Table 5, in which exemplary nucleotide sequences (nt) for the components of the exemplary CAR constructs prior to elimination of the binding site and codon optimization (non-opt), nucleic acid sequences ( nt) for the components of the CAR constructs after elimination and optimization of the binding site (O / SSE), and the corresponding amino acid sequences (aa) encoded by the nucleic acid sequences are provided. Components include the IgG-kappa (ss) signal sequence, the anti-BCMA scFv, spacer region, transmembrane (tm) domain, cosinalization sequence (cossig 4-1BB or cosig CD28), CD3-6 signal domain ( CD3-), ribosomal jump element T2A (T2A) and truncated EGF receptor sequence (EGFRt). Polynucleotide sequences of exemplary CAR constructs are shown in SEQ ID NOs: 751-756, complicating the amino acid sequences mentioned in SEQ ID NOs: 757-762.
[00351] [00351] “Cells are also provided as modified cells that contain a recombinant receptor (for example, a chimeric antigen receptor) as one that contains an extracellular domain including an anti-BCMA antibody or fragment, as described here. Populations of these cells are also provided, compositions containing those cells and / or enriched for those cells, as in which cells expressing the BCMA binding molecule make up at least 50, 60, 70, 80, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or more percent of the total cells in the composition or cells of a particular type, such as T or CD8 + or CD4 + cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Likewise, therapeutic methods are provided to administer cells and compositions to individuals, for example, patients.
[00352] Thus, genetically modified cells are also provided that express the recombinant receptors that contain antibodies, for example, cells that contain CARs. Cells are generally eukaryotic cells, like mammalian cells, and are typically human cells. In some embodiments, cells are derived from blood, bone marrow, lymph or lymphoid organs, are cells of the immune system, such as cells of innate or adaptive immunity, for example, myeloid or lymphoid cells, including lymphocytes, usually T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). Cells are typically primary cells, like those isolated directly from an individual and / or isolated from an individual and frozen. in some embodiments, cells include one or more subsets of T cells or other types of cells, such as entire populations of cells
[00353] [00353] Among the subtypes and subpopulations of T and / or CD4 + and / or CD8 + cells are virgin T (Tn) cells, effector T cells (TerrF), memory T cells and subtypes, such as memory T cells. stem cells (Tscm), central memory T (Tcvm), effective memory T (Tem) or terminally differentiated effector memory T, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells , cytotoxic T cells, mucosal-associated invariant T cells, naturally occurring adaptive and adaptive regulatory T cells (Treg), helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH17 cells, TH9 cells, TH22 cells , follicular helper T cells, alpha / beta T cells and delta / gamma T cells.
[00354] [00354] In some embodiments, the cells are natural killer cells (NK). In some embodiments, the cells are monocytes or granulocytes, for example, myeloid cells, macrophages,
[00355] [00355] In some embodiments, cells include one or more polynucleotides introduced by genetic engineering and, thus, express recombinant or genetically modified products of such polynucleotides. In some embodiments, polynucleotides are heterologous, that is, they are not normally present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which, for example, is not normally found in the cell that is being modified and / or in an organism from which that cell is derived. In some embodiments, polynucleotides do not occur naturally, like a polynucleotide not found in nature, including one that comprises chimeric combinations of polynucleotides that encode several domains of several different cell types. In some embodiments, cells (for example, modified cells) comprise a vector (for example, a viral vector, expression vector, etc.) as described herein, such as a vector comprising a nucleic acid that encodes a recombinant receptor described here. A. Vectors and Methods for Genetic Engineering
[00356] [00356] Methods, polynucleotides, compositions and kits are also provided for expressing binding molecules (eg, anti-BCMA binding molecules), including recombinant receptors (eg, CARs) comprising binding molecules and for produce the genetically modified cells that express these binding molecules. In some embodiments, one or more binding molecules, including recombinant receptors (eg, CARs) can be genetically modified in the cells or plurality of cells. Genetic engineering usually involves introducing a nucleic acid that encodes the recombinant or modified component into the cell, such as by transduction, transfection or retroviral transformation
[00357] [00357] Polynucleotides are also provided that encode the chimeric antigen receptors and / or portions, for example, chains thereof. Among the polynucleotides provided are those that encode the chimeric anti-BCMA antigen receptors (e.g., antigen binding fragment) described herein. Polynucleotides that encode one or more antibodies and / or portions thereof, for example, those that encode one or more of the anti-BCMA antibodies (e.g., antigen binding fragment) described herein and / or other antibodies and / or portions thereof, for example, antibodies and / or portions thereof that bind to other target antigens. Polynucleotides can include those that include nucleotides and natural and / or unnatural bases, for example, including those with modifications in the main chain. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" can be used interchangeably and refer to a nucleotide polymer. Such nucleotide polymers may contain natural and / or unnatural nucleotides and include, however, are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear nucleotide sequence comprising the nucleic acid molecule or polynucleotide.
[00358] [00358] Polynucleotides are also provided that have been optimized for codon use and / or to eliminate binding sites, such as cryptic binding sites. Methods are also provided to optimize and produce the coding sequences for chimeric antigen receptors, like any of the chimeric antigen receptors described here. Such methods are described in Section | on here.
[00359] [00359] “Vectors containing polynucleotides are also provided, like any of the polynucleotides described here, and host cells containing the vectors, for example, to produce
[00360] [00360] “Methods are also provided to manufacture chimeric anti-BCMA antigenic receptors. For recombinant production of chimeric receptors, a nucleic acid sequence encoding a chimeric antibody of the receptor, for example, as described here, can be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell . Such nucleic acid sequences can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to genes that encode the antibody heavy and light chains). In some embodiments, a method is provided to produce the chimeric anti-BCMA antigen receptor, wherein the method comprises culturing a host cell comprising a nucleic acid sequence encoding the antibody, as provided above, under conditions appropriate conditions for receptor expression.
[00361] [00361] In some aspects, for the production of isolated or secreted polypeptides, in addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeasts, are cloning or expression hosts suitable for antibody-encoding vectors, including yeast fungi and strains whose glycosylation pathways have been modified to mimic or approximate human cells, resulting in
[00362] [00362] Exemplary eukaryotic cells that can be used to express polypeptides, including isolated or segregated polypeptides, include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. CHO Lec13 cells and CHO FUT8 cells; PER.C66 cells; and NSO cells. In some modalities, antibody heavy chains and / or light chains (for example, Vx region and / or V region) can be expressed in yeasts. See, for example, North American Publication No. US 2006/0270045 A1. In some embodiments, a specific eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and / or light chains (for example, Vu region and / or V. region). For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
[00363] [00363] In particular examples, immune cells, such as human immune cells, are used to express the polypeptides provided that encode chimeric antigen receptors. In some examples, immune cells are T cells, such as CD4 + and / or CD8 + immune cells, including primary cells, such as primary CD4 + and CD8 + cells.
[00364] [00364] In some modalities, gene transfer is performed by first stimulating the cell, as combining it with a stimulus that induces a response such as proliferation, survival and / or activation, for example, as measured by the expression of a cytokine or activation marker, followed by transduction of activated cells
[00365] [00365] In some contexts, overexpression of a stimulating factor (for example, a lymphokine or a cytokine) can be toxic to an individual. Thus, in some contexts, the modified cells include segments of genes that make the cells susceptible to negative selection in vivo, such as after administration in adoptive immunotherapy. For example, in some ways, cells are modified so that they can be eliminated as a result of a change in the in vivo condition of the patient to whom they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Selectable negative genes include the herpes simplex virus type thymidine kinase gene | (HSV-I TK) (Wigler et al., Cell 2: 223, 1977) which confers sensitivity to ganciclovir; the hypoxanthine cell phosphoribosyltransferase (HPRT) gene, the cell adenine phospho-ribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et a /., Proc. Natl. Acad. Sci. USA 89: 33 (1992)) .
[00366] [00366] In some aspects, cells are modified to promote the expression of cytokines or other factors. Various methods for introducing genetically modified components, for example, antigen receptors, for example, CARs, are well known and can be used with the methods and compositions provided. Exemplary methods include those for transferring polylucleotides that encode receptors, including viral, for example, retroviral or lentiviral, transduction, transposons and electroporation.
[00367] [00367] In some embodiments, recombinant polynucleotides are transferred to cells using particles of recombinant infectious viruses, such as, vectors derived from simian virus 40 (SV40), adenovirus, adenoassociated virus (AAV). In some embodiments, recombinant polynucleotides are transferred to T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 April 3. doi: 10, 1038 / gt, 2014,25; Carlens et al. (2000) Exp Hematol 28 (10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol Trends 29 Biotechnol. November 29, 2011 (11): 550-557).
[00368] [00368] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), for example, a retroviral vector derived from Moloney's murine leukemia virus (MOoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), human immunodeficiency virus type 1 (HIV-1). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any source of avian or mammalian cells. Retroviruses are typically amphotropic, which means that they are capable of infecting host cells of various species, including humans. In one embodiment, the gene to be expressed replaces the retroviral sequences of gag, pol and / or env. A number of illustrative retroviral systems have been described (for example, US patents 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, AD (1990 ) Human Gene Therapy 1: 5-14 Scarpa et al. (1991) Virology 180: 849-852; Burns et a /. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris - Lawrie and Temin (1993) Cur. Opin. Genet. Develop, 3: 102-109.
[00369] [00369] Lentiviral transduction methods are known. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunother. 35 (9): 689-701; Cooper et al. (2003) Blood. 101: 1637-
[00370] [00370] In some embodiments, recombinant polynucleotides are transferred to T cells by electroporation (see, for example, Chicaybam et a /, (2013) PLoS ONE 8 (3): e60298 and Van Teloelo et al. (2000) Gene Therapy 7 (16): 1431-1437). In some embodiments, recombinant polynucleotides are transferred to T cells by transposition (see, for example, Manuri et a /. (2010) Hum Gene Ther 21 (4): 427-437; Sharma et a /. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include transfection of calcium phosphate (for example, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. New York), protoplast fusion, cationic liposome-mediated transfection; bombardment of microparticles facilitated by tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
[00371] [00371] Other methods and vectors for the transfer of polynucleotides that encode recombinant products are those described, for example, in the international patent application, Publication No .: WO2014055668 and United States Patent No. 7,446,190.
[00372] [00372] Among additional polynucleotides, for example, genes for introduction are those that improve the effectiveness of therapy, such as promoting the viability and / or function of the transferred cells; genes to provide a genetic marker for selection and / or evaluation of cells, as well as to assess survival or localization in vivo; genes to improve safety, for example, making the cell susceptible to negative selection in vivo, as described by Lupton S. D. et al. Mol. And Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3:
[00373] [00373] In some embodiments, one or more binding molecules, including antibodies and / or recombinant receptors (eg, CARs), can be genetically modified to be expressed in cells or in the plurality of cells. In some embodiments, a first recombinant receptor and a second binding molecule, for example, recombinant receptor, are encoded by the same or separated nucleic acid molecule. In some modes, additional binding molecules are modified to be expressed in cells or in a plurality of cells.
[00374] [00374] In some cases, the polynucleotide containing nucleic acid sequences that encode the BCMA binding receptor, for example, chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some respects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other respects, the signal sequence can encode a heterologous or non-native signal peptide. In some respects, the exemplary non-limiting signal peptide includes an IgG cap chain signal peptide mentioned in SEQ ID NO: 620, or encoded by the nucleotide sequence mentioned in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain mentioned in SEQ ID NO: 851 and encoded by the nucleotide sequence mentioned in SEQ ID NO: 850; a CD8 alpha signal peptide mentioned in SEQ ID NO: 852; or a CD33 signal peptide mentioned in SEQ ID NO: 853.
[00375] [00375] In some modalities, the vector or construction may con-
[00376] [00376] In some modalities, the vector or construct may contain a single promoter that triggers the expression of one or more nucleic acid molecules. In some embodiments, these nucleic acid molecules, for example, transcriptions, can be multicistronic (bicistronic or tristristronic, see, for example, US Patent No. 6,060,273). For example, in some modalities, the transcription units can be modified as a bicistronic unit containing an IRES (internal ribosome entry site), which allows the coexpression of gene products (for example, encoding a first and a second receptor chimeric) by a message from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (for example, encoding a first and second binding molecules, for example). example, recombinant antibody receptor) separated from each other by sequences encoding a self-cleaving peptide (for example, 2A cleavage sequences) or a protease recognition site (for example, furin). The ORF thus encodes a single polypeptide, which, during (in the case of T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome synthesis) a peptide bond at the C-terminus of an 2A element, leading to the separation between the end of the 2A sequence, and the next downstream peptide (see, for example, Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5: 616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides described herein, without limitation, 2A sequences of foot-and-mouth disease virus (F2A, for example, SEQ ID NO: 659 or 660), equine rhinitis virus (E2A, for example, SEQ ID NO: 657 or 658), Thosea asigna virus (T2A, for example, SEQ ID NO: 631, 653 or 654) and porcine teschovirus-1 (P2A, for example, SEQ ID NO: 655 or 656) as described in U.S. Patent Publication No. 20070116690. In some embodiments, one or more different or separate promoters direct the expression of one or more nucleic acid molecules that encode one or more binding molecules, for example , recombinant receptors.
[00377] [00377] Any of the binding molecules, for example, recombinant antibodies and / or receptors provided herein, for example, BCMA binding molecules and / or additional recombinant receptors, can be encoded by polynucleotides containing one or more nucleic acid molecules that encode the receptors, in any combination or arrangement. For example, one, two, three or more polynucleotides can encode one, two, three or more different receptors or domains. In some embodiments, a vector or construct contains nucleic acid molecules that encode one or more binding molecules, for example, recombinant antibody and / or receptor, and a separate vector or construct contains nucleic acid molecules that encode a additional binding molecule, for example
[00378] [00378] Compositions are also provided that contain one or more of the molecules, vectors or nucleic acid constructs, such as those described above. In some embodiments, nucleic acid molecules, vectors, constructs or compositions can be used to design cells, such as T cells, to express any of the binding molecules, for example, recombinant antibody or receptor and / or the binding molecules additional. B. Preparation of Cells for Modification
[00379] [00379] In some modalities, the preparation of the modified cells includes one or more stages of culture and / or preparation. Cells for introducing the recombinant receptor (for example, CAR) can be isolated from a sample, such as a biological sample, for example, one obtained from or derived from an individual. In some embodiments, the individual from whom the cell is isolated is one who has the disease or condition or needs cell therapy or to whom cell therapy will be administered. The individual in some modalities is a human being needs a specific therapeutic intervention, such as the adoptive cell therapy for which the cells are being isolated, processed and / or modified.
[00380] [00380] Therefore, cells in some embodiments are primary cells, for example, primary human cells. Samples include tissue, fluid and other samples taken directly from the individual, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (eg, transduction with viral vector), washing and / or incubation . The biological sample can be a sample obtained directly from a biological source or a processed sample. Biological samples
[00381] [00381] In some respects, the sample from which the cells are derived or isolated is blood or is a sample derived from blood, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, lymphoid tissue associated with the intestine, lymphoid associated with tissue mucosa, spleen , other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testicles, ovaries, tonsils or other organs and / or cells derived from them. Samples include, in the context of cell therapy, for example, adoptive cell therapy, samples from autologous and allogeneic sources.
[00382] [00382] In some embodiments, cells are derived from | cell lines, for example, T cell lineages. Cells in some embodiments are obtained from a xenogenic source, for example, from mouse, rat, primate not human or pig.
[00383] [00383] In some embodiments, cell isolation includes one or more stages of preparation and / or separation by cells without affinity. In some examples, cells are washed, centrifuged and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for the desired components, lyse or remove cells sensitive to specific reagents. In some examples, cells are separated based on one or more properties, such as density, adherent properties, size, sensitivity and / or resistance to particular components.
[00384] [00384] In some examples, an individual's circulating blood cells are obtained, for example, by apheresis or leukapheresis. The samples, in some respects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets and, in some aspects, contain cells other than red blood cells and platelets.
[00385] [00385] In some embodiments, the blood cells collected from the individual are washed, for example, to remove the plasma fraction and place the cells in a buffer or medium appropriate for the subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some respects, a washing step is performed with a "continuous flow" semi-automated centrifuge (eg, Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ** / Mg ** free PBS. In certain embodiments, the components of a blood cell sample are removed and the cells are resuspended directly in the culture media.
[00386] [00386] In some embodiments, the methods include methods of separating cells based on density, such as preparing white blood cells from peripheral blood, lysing red blood cells and centrifuging through a Percoll or Fi- coll gradient.
[00387] [00387] In some embodiments, the isolation methods include the separation of different types of cells based on the expression or presence in the cell of one or more specific molecules, such as surface markers, for example, surface proteins, intracellular markers or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, separation is a separation based on affinity or immunoaffinity. For example, isolation in some respects includes the separation of cells and cell populations based on the expression or level of expression of the cells of one or more markers, typically cell surface markers, for example, by incubation with a antibody or binding partner that specifically binds to such markers, generally followed by steps of washing and separating cells that have bound to the antibody or binding partner, from those cells that have not bound to the antibody or binding partner.
[00388] [00388] Such separation steps can be based on positive selection, in which cells that bound the reagents are retained for later use and / or negative selection, in which cells that do not bind to the antibody or binding partner are retained . In some instances, both fractions are retained for later use. In some respects, negative selection can be particularly useful when there is no antibody available that specifically identifies a cell type in a heterogeneous population, so that separation is best performed based on markers expressed by cells other than the desired population. .
[00389] [00389] The separation does not need to result in 100% enrichment or removal of a given population of cells or cells that express a particular marker. For example, positive selection or enrichment for cells of a specific type, such as those that express a marker, refers to an increase in the number or percentage of those cells, but does not need to result in a complete absence of cells that do not express the marker. . Likewise, negative selection, removal or depletion of cells of a specific type, such as those that express a marker, refers to the decrease in the number or percentage of these cells, but it does not have to result in the complete removal of all these cells.
[00390] [00390] In some examples, several cycles of separation steps are performed, where the selected fraction positively or negatively from one stage is submitted to another separation stage, as a subsequent positive or negative selection. In some instances, a single separation step can deplete cells that express multiple markers simultaneously, such as incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, several types of cells can be selected positively simultaneously by incubating cells with a plurality of antibodies or binding partners expressed in the various types of cells.
[00391] [00391] For example, in some respects, specific subpopulations of T cells, such as positive cells or expressing high levels of one or more surface markers, for example, CD28 +, CD62L +, CCR7 +, CD27 +, CD127 +, CD4 + T cells , CD8 +, CD45RA + and / or CD45RO + are isolated by positive or negative selection techniques.
[00392] [00392] For example, CD3 +, CD28 + T cells can be selected positively using CD3 / CD28 conjugated magnetic beads (eg, DYNABEADSGO M-450 CD3 / CD28 T Cell Expander DYNABEADSGO M-450, MACSiBeads ”Y , etc.).
[00393] [00393] In some modalities, isolation is carried out by enrichment for a specific cell population by positive selection, or depletion of a specific cell population by negative selection. In some embodiments, positive or negative selection is performed by incubating cells with one or more antibodies or another binding agent that specifically bind to one or more expressed or expressed surface markers (marker *) at a relatively higher level ( marker * * "º) in the positive or negatively selected cells, respectively.
[00394] [00394] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed in non-T cells, such as B cells, monocytes or other white blood cells, such as CD14. In some aspects, the CD4 + and / or CD8 + selection steps are used to separate CD4 + and CD8 + auxiliary cytotoxic T cells from a composition, such as from a PBMC composition, such as that obtained by leukapheresis. Such CD4 + and CD8 + populations, in some respects, can still be classified into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree in one or more subpopulations of virgin T cells, memory and / or effector. In some modalities, the CD4 + and CD8 + cells are mixed in the desired ratio
[00395] [00395] In some modalities, CD8 + cells are further enriched or depleted of virgin stem cells, central memory, effective memory and / or central memory stem cells, as by positive or negative selection based on antigens areas associated with the respective subpopulation. In some modalities, the enrichment of central memory T cells (Tcm) is performed to increase efficiency, as well as to improve long-term survival, expansion and / or graft after administration, which in some aspects is particularly robust in these subpopulations. See Terakura et al. (2012) Blood, 1: 72-82; Wang et al. (2012) J. Immunother. 35 (9): 689-701. In some modalities, the combination of CD8 + T cells enriched with Tcm and CD4 + T cells further improves effectiveness.
[00396] [00396] In the modalities, memory T cells are present in the CD62L + and CD62L- subsets of the CD8 + peripheral blood lymphocytes. PBMC can be enriched or depleted from the CD62L-CD8 + and / or CD62L + CD8 + fractions, such as the use of anti-CD8 and anti-CD62L antibodies.
[00397] [00397] In some embodiments, enrichment for central memory T cells (Tcvm) is based on the positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and / or CD27; in some respects, it is based on negative selection for cells that express or express CD45RA and / or granzyme B. In some respects, the isolation of a CD8 + population enriched for Tem cells is accomplished by depleting cells that express CD4, CD14 , CDA45RA, and positive selection or enrichment for cells that express CD62L. In one respect, enrichment of central memory T cells (Tcm) is accomplished by starting with a negative fraction of cells selected based on the expression of CD4, which is subjected to a negative selection based on the expression of CD14 and CDA45RA and a positive selection based on CD62L. Such selections in some aspects are made simultaneously and in other aspects they are made sequentially, in any order. In some respects, the same selection step based on CD4 expression used in the preparation of the CD8 + cell population or subpopulation is also used to generate the CD4 + cell population or subpopulation, so that the positive and negative fractions separate - CD4-based tion are retained and used in subsequent steps of the methods, optionally following one or more positive or negative selection steps.
[00398] [00398] In a particular example, a sample of PBMCs or other
[00399] [00399] CD4 + T helper cells are classified into virgin, central memory and effector cells, identifying the population of ns cells that have antigens on the cell surface. CD4 + lymphocytes can be obtained by standard methods. In some modalities, virgin CD4 + T lymphocytes are CD45RO-, CD45RAr +, CD62L +, CD4 + cells. In some embodiments, the central memory CD4 + cells are CD62L + and CD45RO +. In some modalities, CD4 + effector cells are CD62L- and CD45RO-.
[00400] [00400] In one example, to enrich CD4 + cells by negative selection, a cocktail of monoclonal antibodies typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some embodiments, the antibody or binding partner is attached to a support or solid matrix, such as a magnetic bead or paramagnetic bead, to allow separation of cells for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic separation (or magnetic affinity) techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Resarch arch Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: SA Brooks and U. Schumacher Humana Press Inc., Totowa, NJ).
[00401] [00401] In some respects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as particles or microparticles responsive to magnetics, such as paramagnetic beads (for example, Dynabeads & or MACSO beads) . Magnetically responsive material, for example, particle, is usually directly or indirectly bound to a binding partner, for example, an antibody that specifically binds to a molecule, for example, surface marker, present in the cell, cells or cell population that he wants to separate, for example, that he wants to select negatively or positively.
[00402] [00402] In some embodiments, the particle or magnetic bead comprises a responsive material magnetically attached to a specific binding member, such as an antibody or other binding partner. There are many known, magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, Pat.
[00403] [00403] Incubation is usually performed under conditions in which antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to these antibodies or binding partners, which are bound to the particle or bead magnetic, surface molecules bind specifically to the cell, if present in the sample cells.
[00404] [00404] In some aspects, the sample is placed in a magnetic field and cells that have magnetically responsive or magnetizable particles attached to it will be attracted to the magnet and separated from unidentified cells. For positive selection, the cells attracted by the magnet are retained; for negative selection, cells that are not attracted (unmarked cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, in which the positive and negative fractions are retained and subsequently processed or subjected to additional separation steps.
[00405] [00405] In certain embodiments, the magnetically responsive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes or streptavidin. In certain embodiments, the magnetic particles are connected to the cells through a coating of primary antibodies specific for one or more markers. In certain embodiments, cells, instead of beads, are marked with a primary antibody or binding partner, and then magnetic particles coated with cell-specific secondary antibody or another binding partner are added ( for example, streptavidin). In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
[00406] [00406] In some embodiments, the magnetically responsive particles are left attached to the cells that must be subsequently incubated, cultured and / or modified; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, magnetic or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles or antibodies conjugated to cleavable ligands, etc. In some embodiments, the magnetic particles are biodegradable.
[00407] [00407] In some modalities, the affinity-based selection is via magnetically activated cell classification (MACSQO) (Miltenyi Biotec, Auburn, CA). The Activated Cell Magnetic Classification (MACSQO) systems are capable of selecting high purity cells with magnetized particles attached to them. In certain modalities, MACSO operates in a way in which non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to the magnetized particles are kept in place while the unbound species are eluted. Then, after the completion of this first elution step, the species that were captured in the magnetic field and prevented from being eluted are released in some way, so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from the heterogeneous cell population.
[00408] [00408] In certain modalities, isolation or separation is performed using a system, device or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture and / or formulation steps . In some respects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user manipulation and / or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009 / 072003 or US 20110003380 A1.
[00409] [00409] In some modalities, the system or device performs one or more, for example, all the stages of isolation, processing, modification and formulation in an integrated or independent system, and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device, which allows the user to program, control, evaluate the result and / or adjust various aspects of processing, isolation, steps modification and formulation.
[00410] [00410] In some aspects, the separation and / or other steps are performed using the CIliniMACSO system (Miltenyi Biotec), for example, for automated separation of cells at the level of clinical scale in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump and several pinch valves. The integrated computer in some aspects controls all the components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a support for the selection column. The peristaltic pump controls the flow throughout the piping set and, together with the pinch valves, ensures the controlled flow of buffer through the system, and the continuous suspension of the cells.
[00411] [00411] The CIliniMACSO system, in some aspects, uses magnetizable particles coupled to antibodies that are supplied in a sterile and non-pyrogenic solution. In some embodiments, after marking the cells with magnetic particles, the cells are washed to remove excess particles. A cell preparation bag is then connected to the set of tubes, which in turn is connected to a bag containing a buffer and a cell collection bag. The tube set consists of pre-assembled sterile tubes, including a pre-column and a separation column, and are for single use only. After the start of the separation program, the system automatically applies the cell sample to the separation column. The marked cells are retained in the column, while the unmarked cells are removed by a series of washing steps. In some embodiments, cell populations for use with the methods described here are not marked and are not retained in the column. In some embodiments, cell populations for use with the methods described here are marked and retained in the column. In some embodiments, cell populations for use with the methods described here are eluted from the column after removing the magnetic field and are collected inside the cell collection bag.
[00412] [00412] In certain modalities, the separation and / or other steps are performed using the CIliniMACS Prodigy & (Miltenyi Bio-tec) system. The CIliniMACS ProdigyO system, in some aspects, is equipped with a cell processing unit that allows automated washing and fractionation of cells by centrifugation. The CIiniMACS Prodigy & system can also include an integrated camera and image recognition software that determines the ideal end point of cell fractionation, discerning the macroscopic layers of the product of the source cell. For example, peripheral blood can be automatically separated into erythrocytes, white blood cells and plasma layers. The CIiniMACS ProdigyO6 system can also include an integrated cell culture chamber that performs cell culture protocols such as cell differentiation and expansion, antigen loading and long-term cell culture. Inlet ports can allow sterile removal, and replenishment of media and cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J. Immunother. 35 (9): 651—660, Terakuraet al. (2012) Blood, 1: 72-82, and Wang et al. (2012) J. Inmunother. 35 (9): 689-701.
[00413] [00413] In some embodiments, a population of cells described here is collected and enriched (or depleted) by flow cytometry, in which cells stained for various cell surface markers are transported in a fluidic stream. In some modalities, a population of cells described here is collected and enriched
[00414] [00414] In some embodiments, antibodies or binding partners are labeled with one or more detectable markers, to facilitate separation for positive and / or negative selection. For example, the separation can be based on binding to fluorescently labeled antibodies. In some examples, the separation of cells based on the binding of antibodies or other specific binding partners to one or more cell surface markers is performed in a fluidic stream, such as by fluorescence-activated cell classification (FACS), including scale preparative (FACS) and / or microelectromechanical system chips (MEMS), for example, in combination with a flow cytometric detection system. Such methods allow positive and negative selection based on several markers simultaneously.
[00415] [00415] In some modalities, the preparation methods include steps for freezing, for example, cryopreservation, the cells, before or after isolation, incubation and / or modification. In some modalities, the subsequent freezing and thawing step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. Any of a variety of solutions and freezing parameters known in some ways can be used. An example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium. This is then diluted 1: 1 with medium, so that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then frozen at -80 ° C. at a rate of 1 º per minute and stored in the vapor phase of a liquid nitrogen storage tank.
[00416] [00416] In some modalities, the methods provided include stages of cultivation, incubation, culture and / or genetic engineering. For example, in some modalities, methods are provided to incubate and / or project depleted cell populations and culture-initiating compositions.
[00417] [00417] Thus, in some embodiments, cell populations are incubated in a culture initiation composition. Incubation and / or modification can be carried out in a culture vessel, such as a unit, chamber, cavity, column, tube, tubing set, valve, flask, culture plate, bag or other vessel for cells. culture or cultivation.
[00418] [00418] In some embodiments, cells are incubated and / or cultured before or in connection with genetic engineering. Incubation steps can include culture, cultivation, stimulation, activation and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include those modified to induce cell proliferation, expansion, activation and / or survival in the population, to mimic exposure to the antigen and / or to prepare the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
[00419] [00419] Conditions may include one or more specific means,
[00420] [00420] In some embodiments, the stimulating conditions or agents include one or more agents, for example, ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent binds or initiates the intracellular signaling cascade of TCOR / CD3 in a T cell. Such agents may include antibodies, such as those specific for a TCR, for example, anti-CD3. In some embodiments, the stimulating conditions include one or more agents, for example ligand, which is capable of stimulating a co-stimulating receptor, for example, anti-CD28. In some embodiments, these agents and / or ligands may be attached to a solid support, such as a codon, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti-CD28 antibody to the culture medium (for example, at a concentration of at least about 0.5 ng / ml). In some embodiments, stimulating agents include IL-2, IL-15 and / or IL-7. In some respects, the IL-2 concentration is at least about 10 units / ml.
[00421] [00421] In some respects, incubation is performed according to techniques such as those described in the United States patent
[00422] [00422] In some embodiments, T cells are expanded by adding to the feed cells of the culture initiator composition, such as undivided peripheral blood mononuclear cells (PBMC) (for example, so that the resulting population of cells contains at least about 5, 10, 20 or 40 or more PBMC feeding cells for each T lymphocyte in the initial population to be expanded); and incubate the culture (for example, long enough to expand the number of T cells). In some respects, non-dividing feeder cells may comprise gamma-irradiated PBMC feeder cells. In some modalities, PBMC is irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some respects, feeder cells are added to the culture medium prior to the addition of T cell populations.
[00423] [00423] In some embodiments, the stimulating conditions included at an appropriate temperature for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees and generally at or about 37 degrees Celsius. Optionally, the incubation may also include the addition of non-divided EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. LCL feeder cells, in some respects, are provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10: 1.
[00424] [00424] In modalities, antigen-specific T cells, such as antigen-specific CD4 + and / or CD8 + T cells, are obtained by stimulating virgin or antigen-specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated for cytomegalovirus antigens, isolating T cells from infected individuals and stimulating cells in vitro with the same antigen.
[00425] [00425] Cells are also provided, such as modified cells, which can bind and / or target various antigens. In some modalities, improved selectivity and specificity are achieved through strategies aimed at multiple antigens. Such strategies generally involve multiple antigen-binding domains, which are normally present in genetically modified antigen receptors and specifically bind to distinct antigen. In some embodiments, cells are modified with the ability to bind more than one antigen. For example, in some embodiments, cells are modified to express multispecific binding molecules. In some embodiments, cells express multiple binding molecules, for example, recombinant receptors, each of which can target an antigen or multiple antigens, for example, a receptor that targets the BCMA, like any described here, and another receptor that targets another antigen, for example, tumor antigen. In some respects, a plurality of genetically modified antigen receptors is introduced into the cell, which specifically binds to different antigens, each expressed in or on the disease or condition to be targeted at the cells or tissues or cells thereof. Such characteristics may, in some respects, address or reduce the likelihood of off-target effects or increase effectiveness. For example, when a single antigen expressed in a disease or condition is also expressed in normal or non-diseased cells, these multiple targeting methods can provide selectivity for the desired cell types, requiring binding via multiple receptors of antigens to activate the cell or induce a specific effective function. In some embodiments, a plurality of cells can be modified to express one or more different binding molecules, for example, recombinant receptors, each of which can target an antigen or multiple antigens.
[00426] [00426] Multispecific cells containing any of the binding molecules described herein are also provided, as cells containing a cell surface protein including anti-BCMA antibody and an additional cell surface protein, as an additional chimeric receptor, which binds to a different antigen or to a different epitope in BCMA. In some embodiments, recombinant receptor expression cell compositions are provided, in which one or more of the binding molecules, multispecific binding molecules and / or recombinant receptors bind and / or target BCMA. In some embodiments, multispecific binding molecules and / or recombinant receptors target one or more different epitopes in BCMA.
[00427] [00427] In some embodiments, cell composition is provided, in which each cell type expresses one or more binding molecules, for example, recombinant receptors. In some modalities, the cell comprises (for example, it has been transformed with) one or more vectors comprising one or more nucleic acids that encode one or more amino acid sequences comprising one or more antibodies and / or portions thereof, for example, its antigen-binding fragments. In some embodiments, one or more of these cells are provided. In some embodiments, a composition containing one or more of these cells is provided. In some embodiments, the one or more cells can express different antibodies, or the same antibody. In some embodiments, each cell expresses one or more antibodies, such as more than one antibody. In some embodiments, each cell expresses a multispecific binding molecule, for example, a multispecific receptor, for example, CAR.
[00428] [00428] In some embodiments, cells include multiple targeting strategies that target BCMA and a second or additional antigen associated with a specific disease or condition. In some embodiments, the second or additional antigen is targeted by a multispecific binding molecule and / or multiple binding molecules and / or a plurality of cells, for example, one or more cells, each modified to express a or more recombinant receptors. In some embodiments, a matching receptor targeting a second or additional antigen is expressed in the same cell as a BCMA-binding molecule or in a different cell.
[00429] [00429] In some modalities, between the second or additional antigens for multiple targeting strategies includes those in which at least one of the antigens is a universal tumor antigen or a family member thereof. In some modalities, the second or additional antigen is an antigen expressed in a tumor. In some embodiments, the BCMA-binding molecules provided here target an antigen on the same type of tumor as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a type of tumor. In some embodiments, the cell further comprises an additional genetically modified antigen receptor that recognizes a second or additional antigen expressed in a disease or condition to be treated and induces a stimulating or activating signal.
[00430] [00430] Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, la, HM1,24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, fibronectin ED-B, an oncogene, an oncogen product
[00431] [00431] In some embodiments, the plurality of antigens, for example, the first antigen, for example, BCMA, and the second or additional antigens, are expressed in the target cell, tissue or disease or condition, as in the cancer cell. In some ways, the cell, tissue, disease, or condition is multiple myeloma or a multiple myeloma cell. One or more of the plurality of antigens is also usually expressed in a cell that it is not desirable to target with cell therapy, such as a normal or unhealthy cell or tissue, and / or the modified cells themselves. In such modalities, by requiring the binding of multiple receptors to obtain a cell response, specificity and / or efficacy is achieved.
[00432] [00432] In some respects, the antigen, for example, the second or additional antigen, such as the disease-specific antigen and / or the related antigen, is expressed in multiple myeloma, as the D member of group 5 of the coupled receptor to G class C protein (GPRC5D), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24), BAFF -R, TACI and / or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, B2-Microglobulin, HM1,24, IGF-1R, IL-6R, TRAIL-R1 , and the type IIA activin receptor (Actria). See, Benson and Byrd, J. Clin. Oncol. (2012) 30 (16): 2013-15; Tao and Anderson, Bone Marrow Research (2011): 924058; Chu et al, Leukemia (2013) 28 (4): 917-27; Garfall et al., Discov Med. (2014) 17 (91): 37-46. In some modalities, antigens include those present in lymphoid tumors, myeloma, AIDS-associated lymphoma and / or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against these antigens are known and include, for example, those described in United States Patent No. 8,153,765; 8,603477, 8,008,450; Pub. North American US20120189622 or US20100260748; and / or International PCT Publications Nos. WO2006099875, WO2Z2009080829 or
[00433] [00433] In some embodiments, cells and methods include multiple targeting strategies, such as expression of two or more genetically engineered receptors in the cell, each recognizing a different antigen and typically each including a different intracellular signaling component . Such multi-directional strategies are described, for example, in International Patent Application, Publication No .: WO 2014055668 A1 (describing combinations of stimulating or activating and co-stimulating CARs, for example, targeting two different antigens present individually outside the target, for example, normal cells, but present only in cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013) (describing cells that express a stimulating or activating and inhibiting CAR, such as those in which the stimulating or activating CAR binds to an antigen expressed on normal or non-sick cells and cells disease or condition to be treated, and the CAR inhibitor binds to another antigen expressed only in normal cells or cells that you do not want to treat).
[00434] [00434] In some embodiments, a plurality of cells, each modified to express one or more recombinant receptors, is provided. For example, in some embodiments, a cell is modified to express a binding molecule that binds and / or targets BCMA, and another cell is modified to express a binding molecule that binds and / or targets a additional or second antigen. In some modalities, cells can express
[00435] [00435] Compositions including BCMA binding molecules, immunoconjugates, recombinant receptors and modified cells are also provided, including pharmaceutical compositions and formulations. Among these compositions are those that include modified cells, such as a plurality of modified cells, expressing the provided anti-BCMA recombinant receptors (for example, CARs).
[00436] [00436] Pharmaceutical formulations are provided comprising recombinant chimeric BCMA-binding antigen receptors or modified cells expressing said receptors, a plurality of modified cells expressing said receptors and / or additional agents for treatment or combination therapy. Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.
[00437] [00437] The term "pharmaceutical formulation" refers to a preparation that is such as to allow the biological activity of an active ingredient contained therein to be effective and that it does not contain additional components that are unacceptably toxic to an individual to which the formulation would be administered.
[00438] [00438] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is not toxic to an individual. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[00439] [00439] In some aspects, the choice of vehicle is determined in part by the particular cell, binding molecule and / or antibody and / or by the method of administration. Therefore, there is a variety of suitable formulations. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some ways, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Vehicles are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to receptors at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octa-decyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzene chloride
[00440] [00440] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st. ed. (May 1, 2005).
[00441] [00441] The formulations of the antibodies described herein can include lyophilized formulations and aqueous solutions.
[00442] [00442] The formulation or composition may also contain more than one active ingredient useful for the indication, disease or particular condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in effective amounts for the intended purpose. Thus, in some embodiments, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, pacituitaxel, rubexit, oxituxelube, rituitxel, rubexit, - be, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, for example, a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids and organic acids, such as tartaric, acetic, citric, mimic, lactic, fumaric, benzoic , glycolic, glyconic, succinic and arylsulfonic acids, for example, p-toluenesulfonic acid.
[00443] [00443] The active ingredients can be captured in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. In certain modalities, the pharmaceutical composition is formulated as an inclusion complex, as a cyclodextrin inclusion complex or as a liposome. Liposomes can serve to target host cells (for example, T cells or NK cells) to specific tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and US Patents
[00444] [00444] The pharmaceutical composition in some aspects may employ release systems over time, delayed release and release.
[00445] [00445] The pharmaceutical composition in some embodiments contains the binding molecules and / or cells in amounts effective to treat or prevent the disease or condition, as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some modalities is monitored by periodic evaluation of the treated individuals. For repeated administrations for several days or more, depending on the condition, treatment is repeated until a desired suppression of the symptoms of the disease occurs. However, other dosing regimens can be useful and can be determined. The desired dosage can be administered by a single bolus administration of the composition, by multiple bolus administrations of the composition or by administration of a continuous infusion of the composition.
[00446] [00446] In certain embodiments, in the context of genetically modified cells that contain the binding molecules, for example, CAR, an individual is administered in the range of about one million to about 100 billion cells, such as, for example , 1 million to about 50 billion cells (for example, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about of 20 billion cells, about 30 billion cells, about 40 billion cells or a range defined by two previous values), such as about 10 to 100 billion cells (for example, about 20 million of cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion of cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells or a range defined by two of the previous values), and in some cases, about 100 million cells to about 50 billion cells (for example, about 120 million cells , about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges and / or that number of cells per kilogram of body weight of the individual. In some aspects, in the context of genetically modified cells that express binding molecules, for example, CAR, a composition may contain at least the number of cells to be administered for a dose of cell therapy, such as about or at least a number of cells described here for administration, for example, in Section VA
[00447] [00447] O can be administered using standard administration techniques, formulations and / or devices. Formulations and devices, such as syringes and vials, are provided for storage and administration of the compositions. The administration of the cells can be autologous or heterologous. For example, immune-responsive cells or progenitors can be obtained from one individual and administered to the same or a different compatible individual. Immunoresponsive cells derived from peripheral blood or its progeny (for example, derived in vivo, ex vivo or in vitro) can be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection or administration parenteral. When administering a therapeutic composition (for example, a pharmaceutical composition containing a genetically modified immune response cell), it will usually be formulated in an injectable unit dosage form (solution, suspension, emulsion).
[00448] [00448] The formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, cell populations are administered parenterally. The term "parenteral", when used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic and intraperitoneal administration. In some embodiments, cell populations are administered to an individual using peripheral systemic administration by intravenous, intraperitoneal or subcutaneous injection.
[00449] [00449] Compositions in some embodiments are supplied as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can in some respects be buffered at a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are a little more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions may comprise vehicles, which may be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol ) and their appropriate mixtures.
[00450] [00450] Sterile injectable solutions can be prepared incorporating
[00451] [00451] Various additives that increase the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers, can be added. The prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable pharmaceutical form can be caused by the use of agents that delay absorption, for example, aluminum monostearate and gelatin.
[00452] [00452] “Extended release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, whose matrices are in the form of molded articles, for example, films or microcapsules.
[00453] [00453] The formulations to be used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.
[00454] [00454] “Pharmaceutical compositions for combination therapy are also provided. Any of the additional agents to have
[00455] [00455] “They also provided methods of use and uses of BCMA-binding molecules, immunoconjugates, recombinant receptors, modified cells and pharmaceutical compositions and formulations thereof, as well as in the treatment of diseases, conditions and disorders in which BCMA is expressed, and / or methods of detection, diagnosis and prognosis. Among such methods, such as treatment methods and uses, are those that involve the administration of cells subjected to modification, such as a plurality of modified cells, expressing the provided anti-BCMA recombinant receptors (for example, CARs). Combination therapy methods are also provided
[00456] [00456] Also provided are methods of administration and uses, such as therapeutic and prophylactic uses, of BCMA binding molecules, including anti-BCMA recombinant receptors (eg CARs), modified cells that express recombinant receptors (e.g., CARs), plurality of cells expressing the receptors and / or compositions comprising them. Such methods and uses include therapeutic methods and uses, for example, involving the administration of molecules (for example, recombinant receptors), cells (for example, modified cells) or compositions containing them, to an individual with a disease, condition - tion or disorder associated with BCMA, such as a disease, condition or disorder associated with BCMA expression, and / or in which cells or tissues express, for example, specifically express BCMA. In some embodiments, the molecule, cell and / or composition is / are administered in an effective amount to effect the treatment of the disease or disorder. Uses of the matching receptors (for example, CARs) and cells (for example, modified cells) in such methods and treatments, and in the preparation of a drug to perform such therapeutic methods are provided here. In some ways, the methods are performed by administering the binding molecules or cells, or compositions comprising them, to the individual who has, has had or suspects having the disease or condition. In some embodiments, the methods thus treat the individual's illness or condition or disorder. Also provided here are the uses of any of the compositions, such as pharmaceutical compositions provided herein, for the treatment of a disease or disorder associated with BCMA, such as use in a treatment regimen.
[00457] [00457] “When used here," treatment "(and grammatical variations thereof, such as" treating "or" treating ") refers to the complete or partial improvement or reduction of a disease or condition or disorder, or a symptom adverse effect or result or phenotype associated with it. Desirable treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression , improvement or palliation of the disease state and improved remission or prognosis. The terms do not imply the complete cure of a disease or the complete elimination of any symptom or effect (s) in all symptoms or results.
[00458] [00458] "When used here," delaying the development of a disease "means postponing, preventing, decreasing, delaying, stabilizing, suppressing and / or postponing the development of the disease (such as cancer). This delay can be of variable duration, depending on the history of the disease and / or the individual to be treated. As a sufficient or significant delay, it can, in effect, encompass prevention, insofar as the individual does not develop the disease. For example, cancer at an advanced stage, such as the development of metastases, can be delayed.
[00459] [00459] "Prevention", when used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual who may be predisposed to the disease, but has not yet been diagnosed with the disease. In some embodiments, the molecules and compositions provided are used to slow the development of a disease or to slow the progression of a disease.
[00460] [00460] “When used here, to" suppress "a function or activity is to reduce the function or activity when compared to the same conditions, except for one condition or parameter of interest, or alternatively, compared to another condition. For example,
[00461] [00461] An "effective amount" of an agent, for example, a pharmaceutical formulation, binding molecule, antibody, cells or composition, in the context of administration, refers to an effective amount, in dosages / quantities and by necessary periods of time to achieve a desired result, such as a therapeutic or prophylactic result.
[00462] [00462] A "therapeutically effective amount" of an agent, for example, a pharmaceutical formulation, binding molecule, antibody, cells or composition refers to an effective amount, in dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for the treatment of a disease, condition or disorder and / or the pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the condition of the disease, age, sex and weight of the individual and the populations of cells administered. In some modalities, the methods provided involve the administration of molecules, antibodies, cells and / or compositions in effective amounts, for example, therapeutically effective amounts.
[00463] [00463] A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in individuals before or in an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
[00464] [00464] “When used here, an" individual "or a" person "is a mammal. In some modalities, a "mammal" includes human beings, non-human primates, domestic and farm and zoo animals, sports or pets, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice , ferrets, rats, cats, monkeys, etc. In some modalities, the individual is human.
[00465] [00465] The methods for administering cells for adoptive cell therapy are known and can be used in connection with the methods and compositions provided. For example, adoptive T cell therapy methods are described, for example, in U.S. Patent Application Publication No. 2003/0170238 by Gruenberg et al; United States Patent No. 4,690,915 to Roseberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10): 577-85). See, for example, Themeli et a /. (2013) Nat Biotechnol. 31 (10): 928-933; Tsu- kharahara et a /. (2013) Biochem Biophys Res Commun 438 (1): 84-9; Da- vila et al. (2013) PLoS ONE 8 (4): e61338.
[00466] [00466] Among the diseases to be treated, is any disease or disorder associated with BCMA or any disease or disorder in which BCMA is specifically expressed and / or in which BCMA has been targeted for treatment (also referred to herein as - interchangeable as a "BCMA-associated disease or disorder"). Cancers associated with BCMA expression include hematological neoplasms, such as multiple myeloma. Waldenstrom's macroglobulinemia, as well as Hodgkin's and non-Hodgkin's lymphomas. See, Coquery et al., Crit Rev Immunol., 2012, 32 (4): 287-305 for a review of the BCMA. Since BCMA is involved in mediating tumor cell survival, it is a potential target for cancer therapy. Chimeric antigenic receptors containing anti-mouse BCMA antibodies and cells that express these chimeric receptors have been described previously. See, Carpenter et al., Clin Cancer Res., 2013, 19 (8): 2048-2060.
[00467] [00467] In some modalities, the disease or disorder associated with BCMA is a B cell related disorder. In some modalities, the disease or disorder associated with BCMA is one or more diseases or conditions among glioblastoma, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunoregulatory disorder, heavy chain disease, primary or associated with immunocytes or monoclonal gammopathy of undetermined significance.
[00468] [00468] In some embodiments, the disease or disorder associated with BCMA is an autoimmune disease or disorder. Such autoimmune diseases or disorders include, but are not limited to, systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis (eg, juvenile rheumatoid arthritis), ANCA-associated vasculitis, thrombocytopenic purpura idiopathic (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, psoriasis, multiple sclerosis IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, phospho-lipid syndrome, Goodpasture's disease, Kawasaki's disease, autoimmune haemolytic anemia, myasthenia gravis or progressive glomerulonephritis.
[00469] [00469] In certain diseases and conditions, BCMA is expressed in malignant cells and cancers. In some modalities, cancer (for example, a BCMA expression cancer) is a B-cell malignancy. In some modalities, cancer (for example, a BCMA expression cancer) is lymphoma, leukemia, or plasma cell malignancy. The lymphomas considered herein include, but are not limited to, Burkitt's lymphoma (for example, endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, follicular lymphoma, lymphoma small non-cleaved cells, lymphoma of linear tissue
[00470] [00470] In some modalities, the methods can identify an individual who has, is suspected of having, or is at risk of developing a disease or disorder associated with BCMA. Therefore, methods are provided to identify individuals with diseases or disorders associated with elevated BCMA expression and select them for treatment with supplied BCMA-binding recombinant receptors (eg, CARs) and / or modified cells that express recombinant receptors.
[00471] [00471] For example, an individual can be screened for the presence of a disease or disorder associated with elevated BCMA expression, such as BCMA expression cancer. In some modes, the methods include tracking or detecting the presence of a disease associated with BCMA, for example a tumor. Thus, in some respects, a sample can be obtained from a patient with a suspected disease or disorder associated with elevated BCMA expression and tested for BCMA expression level. In some respects, an individual who tests positive for a disease or disorder associated with BCMA can be selected for treatment by the present methods and a therapeutically effective amount of a recombinant receptor (for example, CAR) comprising a molecule can be administered. BCMA-binding cells, cells containing a recombinant receptor or their pharmaceutical composition as described herein.
[00472] [00472] In some modalities, the individual has persistent or relapsing disease, for example, after treatment with another BCMA-specific antibody and / or cells that express a chimeric receptor directed to BCMA and / or other therapy, including chemotherapy, radiation and / or hematopoietic stem cell transplants (HSCT), for example, allogeneic HSCT or autologous HSCT. In some modalities, management effectively treats the individual, even though the individual has become resistant to another BCMA-targeted therapy. In some modalities, the individual has not relapsed, but is determined to be at risk of relapse, as at high risk of relapse, and therefore the compound or composition is administered prophylactically, for example, to reduce the likelihood or prevent relapse.
[00473] [00473] In some modalities, the individual is eligible for a transplant, as he is eligible for a hematopoietic stem cell transplant (HSCT), for example, allogeneic HSCT or autologous HSCT. In some of these modalities, the individual has not yet received a transplant, despite being eligible, prior to administration of BCMA-binding molecules, including recombinant anti-BCMA receptors (eg, CARs), modified cells that ex - press recombinant receptors (for example, CARs), plurality of modified cells expressing receptors and / or compositions comprising them, as provided herein.
[00474] [00474] In some modalities, the individual is one who is not eligible for a transplant, as he is not eligible for a hematopoietic stem cell transplant (HSCT), for example, allogeneic HSCT or autologous HSCT. In some of these modalities, this individual is administered to BCMA-binding molecules, including recombinant anti-BCMA receptors (eg, CARs), modified cells that express recombinant receptors (eg, CARs), plurality of modified cells expressing the receptors, and / or compositions comprising the same, according to the modalities provided herein.
[00475] [00475] In some modalities, before the administration of the modified cells started, the individual received one or more previous therapies. In some modalities, the individual received at least 1,2, 3,4,5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more previous therapies. In some modalities, the individual received at least 3, 4, 5, 6, 7, 8, 9, 10 or more previous therapies.
[00476] [00476] In some aspects, the individual relapsed or was refractory to one or more previous therapies. In some respects, previous therapies include treatment with autologous stem cell transplantation (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody; unless the individual was not a candidate or was contraindicated for one or more therapies. And bad-
[00477] [00477] In some embodiments, the method may involve the inclusion or exclusion of specific individuals for therapy with the supplied anti-BCMA antibodies, recombinant receptors and / or cells comprising these receptors, based on criteria, diagnosis or indication specifics. In some modalities, at the time of administration of the cell dose or pre-treatment chemotherapy with lymph nodes, the individual had no active history or plasma cell leukemia (PCL). In some modalities, if the individual had an active or PCL history at the time of administration, the individual may be excluded from being treated according to the methods provided. In some modalities, if the individual develops a PCL, such as a secondary PCL, at the time of administration, the individual may be excluded from being treated according to the methods provided. In some modalities, the assessment of criteria, diagnosis or indication can be performed at the time of screening individuals for eligibility or adequacy of treatment according to the methods provided, at various stages of the treatment regimen, in the time to receive therapy for lymph nodes, and / or immediately before the start of administration of the modified cells or their composition.
[00478] [00478] In some embodiments, treatment does not induce an individual's immune response to therapy and / or does not induce such an response to a degree that prevents effective treatment of the disease or condition. In some respects, the degree of immunogenicity and / or graft response against the host is less than that observed with different, but comparable treatment. For example, in the case of adoptive cell therapy using CAR expression cells, including the anti-BCMA antibodies provided, the degree of immunogenicity and immunogenicity modalities are reduced in comparison with CARs, including a different antibody that binds to a similar epitope, for example, overlapping and / or competing for binding to BCMA with the antibody, such as a mouse or monkey or rabbit or humanized antibody.
[00479] [00479] In some embodiments, the methods include adoptive cell therapy, by which genetically modified cells that express the provided recombinant receptors comprising a BCMA-binding molecule (for example, CARs comprising anti-BCMA antibody or its fragment antigen binding agents) are administered to individuals. This administration can promote cell activation (for example, T cell activation) in a manner directed to BCMA, so that the cells of the disease or disorder are directed to destruction.
[00480] [00480] Thus, the methods and uses provided include methods and uses for adoptive cell therapy. In some embodiments, the methods include administering the cells or a composition that contains the cells to an individual, tissue or cell, such as a person at risk or suspected of having the disease, condition or disorder. In some modalities, cells, populations and compositions are administered to an individual with the specific disease or condition to be treated, for example, via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells or compositions are administered to the individual, as an individual with or at risk for the disease or condition. In some ways, methods like this treat, for example, improving one or more symptoms of the disease or condition, such as decreasing the tumor burden in a BCMA-expressing cancer.
[00481] [00481] The methods for administering cells for adoptive cell therapy are known and can be used in connection with the methods and compositions provided. For example, the methods adopted for T cell therapy are described, for example, in the publication of US patent application No. 2003/0170238 by Gruenberg et al; United States Patent No. 4,690,915 to Roseberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10): 577-85). See, for example, Themeli et a /. (2013) Nat Biotechnol. 31 (10): 928-933; Tsu- kharahara et a /. (2013) Biochem Biophys Res Commun 438 (1): 84-9; Da- vila et al. (2013) PLoS ONE 8 (4): e61338.
[00482] [00482] In some embodiments, cell therapy, for example, adoptive cell therapy, is performed by autologous transfer, in which cells are isolated and / or otherwise prepared from the individual who is to receive cell therapy, or of a sample derived from such an individual. Thus, in some aspects, the cells are derived from an individual, for example, a patient, in need of treatment and the cells, after isolation and processing, are administered to the same individual.
[00483] [00483] In some modalities, cell therapy, for example, adoptive cell therapy, for example, adoptive cell therapy, is performed by allogeneic transfer, in which the cells are isolated and / or prepared from an individual who does not be it an individual who should receive it or who finally receives cell therapy, for example, a first individual. In such embodiments, the cells are then administered to a different individual, for example, a second individual, of the same species. In some modalities, the first
[00484] [00484] In some embodiments, the individual to whom cells, cell populations or compositions are administered is a primate, like a human. In some embodiments, the individual to whom the cells, cell populations or compositions are administered is a non-human primate. In some embodiments, the non-human primate is a monkey (for example, cynomolgus monkey) or an ape. The individual can be male or female and can be of any suitable age, including infant, juvenile, adolescent, adult and geriatric individuals. In some embodiments, the individual is a non-primate mammal, such as a rodent (for example, mouse, rat, etc.). In some examples, the patient or individual is an animal model validated for disease, adoptive cell therapy and / or for assessing toxic results, such as cytokine release syndrome (CRS).
[00485] [00485] BCMA-binding molecules, such as matching receptors (eg CARs) and cells expressing the same, can be administered by any suitable means, for example, by injection, for example, intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intracoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, peribulbar injection or release posterior juxtascleral. In some modalities, they are administered by parenteral, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic or subcutaneous administration. Of-
[00486] [00486] For the prevention or treatment of the disease, the appropriate dosage of the binding molecule, the recombinant recipient cell may depend on the type of disease to be treated, the type of binding molecule or recombinant receptor, the severity and course of the disease , whether binding to the molecule's recombinant receptor or is administered for preventive or therapeutic purposes, prior therapy, patient's medical history and response to the recombinant receptor or cell, and at the discretion of the attending physician. The compositions and molecules and cells are, in some modalities, administered properly to the patient at the same time or during a series of treatments.
[00487] [00487] In some modalities, the dose and / or frequency of administration is determined based on effectiveness and / or response. In some modalities, effectiveness is determined by assessing the status of the disease. Exemplary methods for assessing disease status include: measurement of protein M in biological fluids, such as blood and / or urine, by electrophoresis and immunofixation; quantification of sFLC (K and A) in the blood; skeletal research and positron emission tomography (PET) / computed tomography (CT) imaging in individuals with extramedullary disease. In some modalities, the status of the disease can be assessed by examining the bone marrow. In some instances, the dose and / or frequency of administration is determined by the expansion and persistence of the recombinant receptor or cell in the blood and / or bone marrow. In some modalities, the dose and / or frequency of administration is determined based on the anti-tumor activity of the recombinant receptor or modified cell. In some modalities, antitumor activity is determined by the response rate
[00488] [00488] In some modalities, the disease or disorder to be treated is multiple myeloma. In some modalities, measurable disease criteria for multiple myeloma may include (1) serum M protein 1 g / dL or greater; (2) M protein in urine 200 mg or more / 24 hours; (3) serum free light chain involved (sSFLC) 10 mg / dL or greater, with an abnormal K / A ratio. In some cases, light chain disease is acceptable only to individuals without measurable disease in the serum or urine.
[00489] [00489] In some modalities, the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select individuals for treatment, for example, individuals who have performed poorly on previous therapies (see , for example, Oken et al. (1982) J Clin Oncol. 5: 649-655). The Performance Status ECOG Scale describes a patient's level of functioning in terms of their ability to take care of themselves, daily activity and physical ability (eg, walking, working, etc.). In some modalities, an ECOG performance status of 0 indicates that an individual can perform normal activity. In some aspects, individuals with ECOG performance status equal to 1 exhibit some restriction in physical activity, but the individual is totally outpatient. In some respects, patients with an ECOG performance status of 2 are more than 50% outpatient. In some cases, the individual with an ECOG performance status of 2 may also be able to take care of themselves; see, for example, Sgrensen et a /., (1993) Br J. Cancer 67 (4) 773-775. In some embodiments, the individual to be administered according to the methods or treatment regimen provided herein includes those with an ECOG performance status of O or 1.
[00490] [00490] In some modalities, the administration can treat the individual, although the individual has become resistant to another therapy. In some modalities, when administered to individuals according to the modalities described here, the dose or composition is able to achieve an objective response (OR), in at least 50%, 60%, 70%, 80% , 90% or 95% of individuals who were administered. In some modalities, OR includes individuals who achieve strict complete response (sSCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimal response (MR). In some modalities, when administered to individuals in accordance with the modalities described here, the dose or composition is capable of obtaining a strict complete response (sCR), complete response (CR), very good partial response (VGPR) or partial response (PR ), in at least 50%, 60%, 70%, 80% or 85% of the individuals who were administered. In some modalities, when administered to individuals in accordance with the modalities described here, the dose or
[00491] [00491] In some modalities, the toxicity and / or side effects of the treatment can be monitored and used to adjust the dose and / or the frequency of administration of the recombinant receptor, for example, CAR, cells and / or compositions. For example, adverse events and laboratory abnormalities can be monitored and used to adjust the dose and / or the frequency of administration. Adverse events include reactions to infusion, cytokine release syndrome (CRS), neurotoxicity, macrophage activation syndrome and tumor lysis syndrome (TLS). Any of these events can establish dose-limiting toxicities and justify lowering the dose and / or ending treatment. Other side effects or adverse events that can be used as a guide to establish the dose and / or frequency of administration include non-hematological adverse events, which include, among others, fatigue, fever or febrile neutrality, increased transaminases due to a specified period (for example, less than or equal to 2 weeks or less than or equal to 7 days), headache, bone pain, hypotension, hypoxia, chills, diarrhea, nausea / vomiting, neurotoxicity (for example, confusion, aphasia, epileptic seizures, seizures, lethargy and / or altered mental status), coagulation
[00492] [00492] In some modalities, treatment according to the methods provided may result in a lower rate and / or a lower degree of toxicity, toxic result or symptom, profile, factor or toxicity-promoting property, such as a symptom or result associated or indicative of cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, for example, compared to the administration of other therapies.
[00493] [00493] In certain embodiments, in the context of genetically modified cells containing the binding molecules or recombinant receptors, a range of about one million to about 100 billion cells and / or the amount of of cells per kilogram of body weight, for example, about 1 million to about 50 billion cells (for example, about 5 million cells, about 25 million cells, about 500 million cells , about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by two of the previous values), such as about 10 to 100 billion cells (for example, about 20 million cells, about 25 million cells, about 30 million cells, about 40 million cells, about 50 million cells, about 60 million cells cells, about 70 million cells, about 80 million cells cells, about 90 million cells, about 10 billion cells, about 25 billion cells
[00494] [00494] In some embodiments, the methods comprise administering a dose of the modified cells or a composition comprising a dose of the modified cells. In some embodiments, the modified cells or compositions containing modified cells can be used in a treatment regimen, wherein the treatment regimen comprises administering a dose of the modified cells or a composition comprising a dose of the modified cells. In some embodiments, the dose may contain, for example, a particular number or range of recombinant receptor expression T cells, total T cells or total peripheral blood mononuclear cells (PBMCs), like any number of such cells described herein. In some embodiments, a composition containing a dose of the cells can be administered. In some aspects, the number, quantity or ratio of
[00495] [00495] In some modalities, for example, where the individual is human, the dose includes more than about 1 x 10th total recombinant receptor expression cells (eg CAR), T cells or peripheral blood mononuclear cells (PBMCs) and less than about 2 x 10 th total recombinant receptor expression cells (eg CAR), T cells or peripheral blood mononuclear cells (PBMCs), for example, in the range of about 2.5 x 107 about 1.2 x 10º of these cells, such as 2.5 x 107, 5x 107, 1.5 x 108, 3 x 108, 4.5 x 108, 8 x 10º or 1.2 x 10º of total cells, or the range between any two of the previous values.
[00496] [00496] In some embodiments, the dose of genetically modified cells comprises between about 2.5 x 107 T cells of expression of CAR, total T cells or peripheral whole blood mononuclear cells (PBMCs) and about 1 , 2 x 10 th CAR expression T cells, total T cells or total PBMCs, between, about 5.0 x 107 CAR expression T cells and a or about 4.5 x 10 th T cells of ex - CAR pressure, total T cells or total peripheral blood mononuclear cells (PBMCs), between or about 1.5 x 10º T cells of CAR expression and a or about 3.0 x 10º T cells of expression of CAR, total T cells or total PBMCs, each inclusive. In some embodiments, the number refers to the total number of CD3 + or CD8 +, in some cases also CAR expression cells (for example, CAR +). In some embodiments, the dose comprises a number of cells of either about 2.5 x 107 to or about 1.2 x 10 th total CD3 + or CD8 + T cells or CD3 + or CD8 + CAR expression cells, of or about 5.0 x 107 to or about 4.5 x 10º total CD3 + or CD8 + T cells or CD3 + or CDB8 + CAR expression cells, or about 1.5 x 10º to or about 3.0 x 10th total CD3 + or CD8 + T cells expressing CD3 + or CD8 + CAR expression cells, each inclusive.
[00497] [00497] In some embodiments, dose T cells include CD4 + T cells, CD8 + T cells or CD4 + and CD8 + T cells.
[00498] [00498] In some embodiments, for example, where the individual is human, the CD8 + T cells in the dose, including in a dose including CD4 + and CD8 + T cells, include between about 1 x 10 th and a or about 2 x 10 th cells Total recombinant receptor expression CD8 + (eg, CAR), for example, in the range of about 5 x 107 to about 4.5 x 10 th cells, such as or about 2.5 x 107, to about 5 x 107, at or about 1.5 x 108, at or about 3 x 108, at or about 4.5 x 108, at or about 8 x 10º, or at or about 1.2 x 10º total of those cells, or the range between any two of the previous values.
[00499] [00499] In some embodiments, the cell dose, for example, recombinant receptor expression T cells, is administered to the individual as a single dose or is administered only once within a period of two weeks, one month, three months , six months, | year or more. In some modalities, the patient receives multiple doses and each dose or the total dose can be within any of the previous values. In some embodiments, cells modified for administration or composition of cells modified for administration exhibit properties indicative of or consistent with the health of the cells. In some embodiments, the or about or at least the or about 70, 75, 80, 85 or 90% CAR + cells of that dose exhibit one or more properties or phenotypes indicative of cell health or biologically active CAR cell, as an expression absence of an apoptotic marker.
[00500] [00500] In particular modalities, the phenotype is or includes an absence of apoptosis and / or an indication that the cell is going through the apoptotic process. Apoptosis is a programmed cell death process that includes a series of stereotyped morphological and biochemical events that lead to characteristic cell changes and death, including flictin formation, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal fragmentation of the DNA and global deterioration of mMRNA. In some aspects, the early stages of apoptosis can be indicated by the activation of certain caspases, for example, 2, 8, 9 and 10. In some aspects, the middle and late stages of apoptosis are characterized by loss Additional membrane integrity, chromate condensation and DNA fragmentation include biochemical events, such as the activation of caspases 3, 6 and 7.
[00501] [00501] In particular modalities, the phenotype is a negative expression of one or more factors associated with programmed cell death, for example, pro-apoptotic factors known to initiate apoptosis, for example, members of the death receptor pathway, limbs activated from the mitochondrial pathway (intrinsic), as members of the Bcl-2 family, for example, Bax, Bad and Bid and caspases. In certain modalities, the phenotype is the absence of an indicator, for example, an annexin V molecule or by staining with TUNEL, which will preferentially bind to cells subjected to apoptosis when incubated with or contacted with a cell composition. In some embodiments, the phenotype is or includes an expression of one or more markers that are indicative of an apoptotic state in the cell. In some modalities, the phenotype is the lack of expression and / or activation of a caspase, such as caspase 3. In some aspects, the activation of caspase-3 is indicative of an increase or rebirth of apoptosis. In certain embodiments, activation of caspase can be detected by known methods. In some embodiments, an antibody that specifically binds to an activated caspase (that is, it specifically binds to the cleaved polypeptide) can be used to detect caspase activation. In particular modalities, the phenotype is or includes an active 3- caspase. In some modalities, the apoptosis marker is a reagent that detects a resource in a cell that is associated with apoptosis. In certain embodiments, the reagent is an annexin V molecule.
[00502] [00502] In some embodiments, the compositions containing the modified cells for administration contain a certain number or quantity of cells that exhibit phenotypes indicative or consistent with the health of the cells. In some modalities, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the T cells of expression of CAR in the dose of modified T cells express an apoptosis marker, optionally annexin V or active Caspase 3. In some modalities, less than 5%, 4%, 3%, 2% or 1% of the expression T cells of CAR in the dose of modified T cells express annexin V or active Caspase 3.
[00503] [00503] In some embodiments, the cells administered are immune cells modified to express the BCMA-binding recombinant receptor, for example, CAR. In some embodiments, the immune cells are T cells. In some embodiments, the cells administered are CD4 + T cells. In some modalities, the cells administered are CD8 + T cells. In some embodiments, the cells administered are a combination of CD4 + and CD8 + T cells, as a combination of CAR T CD4 + cells and CAR T CD8 + cells, which in some respects are within the same vessel or cell composition or suspension. In some instances, the ratio of CD4 + cells to CD8 + cells (CD4: CD8) administered, such as
[00504] [00504] In some embodiments, cells, binding molecules or recombinant receptors are administered as part of a combination treatment, as simultaneously or sequentially with, in any order, another therapeutic intervention, such as another antibody or cell, receptor or modified agent, such as a cytotoxic or therapeutic agent.
[00505] [00505] Cells, binding molecules and / or recombinant receptors in some modalities are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, simultaneously or sequentially in any order. In some contexts, cells are co-administered with another therapy sufficiently close in time, so that cell populations increase the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells,
[00506] [00506] In some modalities, the individual may receive bridge therapy after leukapheresis and before chemotherapy with lymphedema. An attending physician can determine whether bridge therapy is necessary, for example, for disease control, during the manufacture of the composition or cells supplied. In some embodiments, bridge therapies do not include biological agents, such as antibodies (eg, daratumumab). In some modalities, point therapies are discontinued before the beginning of lympho-exhaustion. In some modalities, bridging therapies are discontinued 1 day, 2 days 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 45 days or 60 days before lymphatic exhaustion.
[00507] [00507] “Once the cells are administered to a mammal (for example, a human), the biological activity of the modified cell populations and / or antibodies in some ways is measured by any of several known methods. The parameters to be evaluated include the specific binding of a modified or natural T cell or another cell immune to the antigen, in vivo, for example, by image or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the modified cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32 (7) : 689-702 (2009) and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004). In certain modalities, the biological activity of cells can also be measured by testing the expression and / or secretion of certain cytokines, such as CD 107a, IFNy, I1L-2 and TNF. In some aspects, biological activity is measured by assessing the clinical outcome, such as a reduction in tumor burden or burden.
[00508] [00508] In certain modalities, the modified cells are modified in several ways, so that their therapeutic or prophylactic efficacy is increased. For example, the modified CAR or TCR expressed by the population in some modalities is conjugated directly or indirectly through a linker to a fraction of targeting. The practice of conjugating compounds, for example, CAR or TCR, to target moieties is known in the art. See, for example, Wadwa et al., J. Drug Targeting, 3 (2): 111 (1995), and United States Patent
[00509] [00509] “Combination therapy methods are also provided that include the administration and uses, such as therapeutic and prophylactic uses, of BCMA-binding recombinant receptors (for example, CARs), modified cells that express recombinant receptors - binant (for example, CARs), plurality of modified cells that express the receptors and / or compositions comprising them.
[00510] [00510] In some embodiments, the BCMA-binding recombinant receptor (eg, chimeric antigen receptor) and / or modified cells expressing said molecules (eg, recombinant receptor) described herein are administered as part of a combination treatment or combination therapy, as simultaneously with, sequentially or intermittently with, in any order, one or more additional therapeutic interventions. In some embodiments, one or more additional therapeutic interventions include, for example, an antibody, a modified cell, a receptor and / or an agent, such as a cell that expresses a response.
[00511] [00511] In some embodiments, the additional agent for combination treatment or combination therapy enhances, increases and / or promotes the efficacy and / or safety of the therapeutic effect of binding molecules, recombinant receptors, cells and / or compositions. In some embodiments, the additional agent increases or improves the efficiency, survival or persistence of the administered cells, for example, cells that express the binding molecule or a recombinant receptor. In some embodiments, the additional agent is selected from a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an immunomodulator or an agent that decreases the level or activity of a regulatory T cell (Treg). In some modalities, the additional agent increases safety, due to the reduction or improvement of the adverse effects of the administered binding molecules, receptors, cells and / or recombinant compositions. In some modalities, the additional agent can treat the same disease, condition or comorbidity. In some embodiments, the additional agent can improve, reduce or eliminate one or more toxicities, adverse effects or side effects that are associated with the administration of recombinant receptors, cells and / or compositions, for example, CAR expression cells.
[00512] [00512] In some modalities, pain control medications, such as acetaminophen or antihistamine, such as diphenhydramine, can be administered before, during or after the administration of the receptor, cell or recombinant composition provided here, to improve improve or reduce or eliminate minor side effects associated with treatment. In some instances, transfusions of red blood cells and platelets and / or colony stimulating factors may be administered to reduce or eliminate one or more toxicities, adverse effects or side effects associated with the administration of receptors, cells and / or recombinant compositions, for example, CAR expression cells. In some modalities, anti-infective agents - prophylactic or empirical (eg, trimethoprim / sulfamethoxazole for prophylaxis for pneumocystis pneumonia [PCP], broad-spectrum antibiotics, antifungals or antiviral agents for febrile neutropenia) may be administered to treat side effects resulting from treatment. In some instances, when necessary, prophylaxis may be provided to treat lymphopenia and / or neutropenia that occurs as a result of treatment.
[00513] [00513] In some embodiments, the therapy, treatment or additional agent includes chemotherapy, radiotherapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth-inhibiting agents, anti-hormonal agents, kinase inhibitors, antiangiogenic agents, cardioprotectors, immunostimulating agents, immunosuppressive agents,
[00514] [00514] In some embodiments, BCMA-binding recombinant cells, receptors and / or compositions, for example, CAR expression cells, are administered in combination with other modified cells, for example, other expression cells CAR. In some embodiments, the additional agent is a kinase inhibitor, for example, a Bruton tyrosine kinase (Btk) inhibitor, for example, ibrutinib. In some embodiments, the additional agent is an adenosine pathway or an adenosine receptor antagonist or agonist. In some embodiments, the additional agent is an immunomodulator, such as thalidomide or a thalidomide derivative (for example, lenalidomide). In some embodiments, the additional agent is a gamma secretase inhibitor, such as a gamma secretase inhibitor that inhibits or reduces the intramembrane cleavage of a gamma secretase target, for example, BCMA, in a cell (such as a cell tumor / cancer). In some embodiments, the additional therapy, agent or treatment is a cytotoxic or chemotherapeutic agent, a biological therapy (for example, antibody, for example, monoclonal antibody or cell therapy) or an inhibitor (for example, kinase inhibitor).
[00515] [00515] In some embodiments, the additional agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (for example, doxorubicin, such as liposomal doxorubicin); an alkaloid vinca (for example, vinblastine, vincristine, vindesine, vinorelbine); an alkylating agent (for example, cyclophosphamide, decarbazazine, melphalan, ifosfamide, temozolomide); an immune cell antibody (for example, alentuzumab, gentuzumab, rituximab, tositumomab); an antimetabolite (including, for example, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, such as fludarabine); a TNFR-related protein agonist (GITR) induced by TNFR glyco-corticosteroids; a proteasome inhibitor (for example, aclacinicin A, gliotoxin or bortezomib); an immunomodulator, such as thalidomide or a thalidomide derivative (for example, lenalidomide).
[00516] [00516] In some modalities, the therapy or additional treatment is cell therapy, for example, adoptive cell therapy. In some embodiments, additional therapy includes administration of modified cells, for example, additional CAR expression cell. In some embodiments, the additional modified cell is a CAR expression cell, which expresses the same or different recombinant receptor as the modified cells provided herein, for example, anti-BCMA CAR expression cells. In some embodiments, the recombinant receptor, for example, CAR, expressed in the additional modified cell, recognizes a different antigen and / or epitope. In some embodiments, the recombinant receptor, for example, CAR, expressed in the additional modified cell, recognizes an epitope different from the same antigen as the recombinant receptors described herein, for example, BCMA. In some embodiments, the recombinant receptor, for example, CAR, expressed in the additional modified cell, recognizes a different antigen, for example, a different tumor antigen or a combination of antigens. For example, in some embodiments, the recombinant receptor, for example, CAR, expressed in the additional modified cell, targets cancer cells that express early lineage markers, for example, cancer stem cells, while other CAR expression cells target cells carcinogens that express posterior lineage markers. In such embodiments, the additional modified cell is administered before, simultaneously, or after administration (e.g., infusion) of the CAR expression cells described herein. In some embodiments, the additional modified cell expresses allogeneic CAR.
[00517] [00517] In some embodiments, the configurations of one or more of the CAR molecules comprise a primary intracellular signaling domain and two or more, for example, 2, 3, 4 or 5 or more, co-stimulating signaling domains. In some embodiments, one or more of the CAR molecules may have the same or a different primary intracellular signaling domain, the same or different
[00518] [00518] In some embodiments, the additional agent is any of the cells modified to express one or more of the anti-BCMA binding molecules and / or cells modified to express additional binding molecules, for example, recombinant receptors, for example, CAR, which target a different antigen. In some embodiments, the additional agent includes any of the cells or plurality of cells described herein, for example, in Section L1.C. In some embodiments, the additional agent is a cell modified to express a recombinant receptor, for example, CAR, targeting a different epitope and / or antigen, for example, a different antigen associated with a disease or condition. In some modalities, the additional agent is a cell modified to express a recombinant receptor, for example, CAR, targeting a second or additional antigen expressed in multiple myeloma, for example, CD38, CD138, CS-1, BAFF-R, TACI and / or FcRH5.
[00519] [00519] In some embodiments, the additional agent is an immunomodulatory agent. In some embodiments, the combination therapy includes an immunomodulatory agent that can stimulate, amplify and / or increase the antitumor immune response, for example the antitumor immune response of the modified cells administered, such as inhibiting immunosuppressive signaling or improving immunostimulatory signaling. In some embodiments, the immunomodulatory agent is a peptide, protein, or small molecule. In some embodiments, the protein may be a fusion protein or a recombinant protein. In some embodiments, the immunomodulatory agent binds to an immunological target, such as a cell surface receptor expressed on immune cells, such as T cells, B cells or antigen presenting cells. For example, in some embodiments, the immunomodulatory agent is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide. In some embodiments, the recombinant receptors, cells and / or compositions are administered in combination with an additional agent that is an antibody or its antigen-binding fragment, such as a monoclonal antibody.
[00520] [00520] In some embodiments, the immunomodulating agent blocks, inhibits or neutralizes a component of the immune checkpoint pathway. The immune system has several inhibitory pathways involved in maintaining self-tolerance and in modulating immune responses. Tumors can use certain immune checkpoint pathways as an important mechanism of immune resistance, particularly against T cells that are specific for tumor antigens (Pardoll (2012) Nature Reviews Cancer 12: 252-264), for example, cells modified as CAR expression cells. Since many of these immune checkpoints are initiated by γ-receptor interactions, they can be easily blocked by antibodies against the ligands and / or their receptors.
[00521] [00521] - Therefore, therapy with antagonistic molecules that block an immune checkpoint pathway, such as small molecules, nucleic acid inhibitors (eg, RNAi) or antibody molecules, are becoming promising avenues for immunotherapy for cancer and other diseases. In contrast to most anticancer agents, checkpoint inhibitors do not necessarily
[00522] [00522] - When used here, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere or modulate one or more checkpoint proteins. Checkpoint proteins regulate T cell activation or function. These proteins are responsible for co-stimulating or inhibiting T cell responses. Immune checkpoint proteins regulate and maintain self-tolerance, and duration and amplitude of physiological immune responses. In some embodiments, the individual may be administered an additional agent that can improve or enhance the immune response, for example, immune response by the BCMA-binding recombinant receptors, cells and / or compositions provided here, against a disease or condition, for example. example, a cancer, like any one described here.
[00523] [00523] Immune checkpoint inhibitors include any agent that blocks or inhibits, in a statistically significant way, the inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies or their antigen-binding fragments, which bind to and block or inhibit immune checkpoint receptors, ligands and / or receptor-ligand interaction. In some modalities, modulation, enhancement and / or stimulation of specific receptors can overcome the components of the immune checkpoint pathway. Illustrative immune checkpoint molecules that can be targeted for blocking, inhibition, modulation, enhancement and / or stimulation include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA4, LAG-3 (CD223), TIM-3, 4-1BB
[00524] [00524] Exemplary immune checkpoint inhibitors include Tremelimumab (CTLA-44 blocking antibody, also known as ticilimumab, CP-675,206), anti-ti-OX40 monoclonal antibody, PD-L1 (Anti-B7-H1; MEDI4736 ), MK-3475 (PD-1 blocker)), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), monoclonal antibody BY55, AMP224 (anti-PD-L1 antibody), BMS- 936559 (anti-PD-L1 antibody), MPLDL3280A (anti-PD-L1 antibody), MSBO0010718C (anti-PD-L1 antibody) and ipilimumab (anti-CTLAH antibody, also known as YervoyO, MDX-010 and MDX- 101). Exemplary immunomodulatory antibodies include, but are not limited to, Daclizumab (Zenapax), Bevacizumab (Avastin O), Basiliximab, Ipilimumab, Nivolumab, Pembrolizumab, MPDL3280A, Pidilizumabe (CT-011), MK-3475, MK-3475, MK-3475, MK-3475 , tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA- CD40, CP-870, CP-893, MEDI6469, MEDI669 , MOXRO0916, AMP-
[00525] [00525] Programmed cell death 1 (PD-1) is an immune checkpoint protein that is expressed in B cells, NK cells and T cells (Shinohara et a /., 1995, Genomics 23: 704-6; Blank et al., 2007, Cancer Immunol Immunother 56: 739-45; Finger et a /., 1997, Gene 197: 177-87; Pardoll (2012) Nature Reviews Cancer 12: 252-264). The primary role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity. PD-1 expression is induced in activated T cells, and binding of PD-1 to one of its endogenous ligands acts to inhibit T cell activation by inhibiting stimulating kinases. PD-1 also acts to inhibit the "interruption signal" from the TCR. PD-1 is highly expressed in Treg cells and can increase its proliferation in the presence of a ligand (Pardoll (2012) Nature Magazine Cancer 12: 252-264). Anti-PD 1 antibodies have been used to treat melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple negative breast cancer , leukemia, lymphoma and renal cell cancer (Topalian et a /., 2012, N Engl J Med 366: 2443-54; Lipson et a /., 2013, Clin Cancer Res 19: 462-8; Berger et al., 2008, Clin Cancer Res 14: 3044-51; Gildener- Leapman et al.,
[00526] [00526] PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 and B7-DC) are ligands for PD-1, found in activated T cells, B cells, myeloid cells, ma - crophages and some types of tumor cells. Anti-tumor therapies focused on anti-PD-L1 antibodies. The PD-1 and PD-L1 complex inhibits the proliferation of CD8 + T cells and reduces the immune response (Topalian et a /., 2012, N Engl J Med 366: 2443-54; Brahmer et al.,
[00527] [00527] The cytotoxic antigen associated with T lymphocytes (CTLA-4), also known as CD152, is a co-inhibitory molecule that works to regulate the activation of T cells. CTLA-4 is a member of the immunoglobulin superfamily that is expressed exclusively in T cells. CTLA-H acts to inhibit T cell activation and is reported to inhibit the activity of helper T cells and to improve the regulatory immunosuppressive activity of T cells. Although the precise mechanism of action of CTLA-4 remains under investigation, it has been suggested that it inhibits T cell activation, overcoming CD28 in binding to CD80 and CD86, in addition to actively providing inhibitory signals to the T cell (Pardoll (2012) Nature Reviews Cancer 12: 252- 264). Anti-CTLA-4 antibodies have been used in clinical trials for the treatment of melanoma, prostate cancer, small cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14 : 848-61; Ott et al. 2013, Clin Cancer Res 19: 5300; Weber, 2007, Oncologist 12: 864-72; Wada et al., 2013, J Transl Med 11:89). A significant feature of anti-CTLA + is the kinetics of the antitumor effect, with a delay period of up to 6 months after the initial treatment required for the physiological response. In some cases, tumors may actually increase in size after starting treatment, before a reduction is observed (Pardoll (2012) Nature Reviews Cancer 12: 252-264). Exemplary anti-cCTLA4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab has recently received FDA approval for the treatment of metastatic melanoma (Wada et al., 2013, J Trans! Med 11:89).
[00528] [00528] The lymphocyte activation gene 3 (LAG-3), also known as CD223, is another protein of the immune checkpoint. LAG-3 has been associated with inhibition of lymphocytic activity and, in some cases, with the induction of lymphocytic anergy. LAG-3 is expressed in several cells of the immune system, including B cells, NK cells and dendritic cells. LAG-3 is a natural ligand for the MHC class II receptor, which is substantially expressed in melanoma infiltrating T cells, including those with potent immunosuppressive activity. Exemplary anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers Squib), which is a monoclonal antibody that targets LAG-3. IMP701 (Immutep) is an antagonist LAG-3 antibody, and IMP731 (Immutep and GlaxoSmithKline) is a depleted LAG-3 antibody. Other inhibitors of LAG-3 include IMP321 (Immutep), which is a recombinant fusion protein of a soluble portion of LAG-3 and lg that binds to MHC class molecules | and activates antigen presenting cells (APC). Other antibodies are described, for example, in WO2010 / 019570 and US 2015/0259420
[00529] [00529] - The immunoglobulin domain of T cells, and the mucin-3 domain (TIM-3), initially identified in activated Th1 cells, proved to be a negative regulator of the immune response. The blocking of TIM-3 promotes T cell-mediated anti-tumor immunity and has anti-tumor activity in several mouse tumor models. Combinations of TIM-3 blockade with other immunotherapeutic agents, such as TSR-042, anti-CD137 antibodies and others, can be additive or synergistic in increasing antitumor effects. TIM-3 expression has been associated with several different types of tumors, including melanoma, NSCLC and kidney cancer. In addition, the expression of intratumoral TIM-3 has shown a correlation with poor prognosis in a variety of types of tumor, including NSCLC, cervical, and gastric cancers. Blocking TIM-3 is also interested in promoting greater immunity to various chronic viral diseases. It has also been shown that TIM-3 interacts with several ligands, including galectin-9, phosphatidylserine and HMGB1, although which one, if any, is relevant in regulating antitumor responses, is not yet clear at the moment . In some modalities, antibodies, antibody fragments, small molecules or peptide inhibitors that target TIM-3 can bind to the IgV domain of TIM-3 to inhibit interaction with its ligands. Exemplary antibodies and peptides that inhibit TIM-3 are described in US 2015/0218274, WOZ2013 / 006490 and US 2010/0247521. Other anti-TIM-3 antibodies include humanized versions of RMT3-23 (Ngiow et a / l., 2011, Cancer Res, 71: 3540-3551), and clone 8B, 2C12 (Monney et a / l., 2002, Nature, 415: 536-541). Bispecific antibodies that inhibit TIM-3 and PD-1 are described in US 2013/0156774.
[00530] [00530] In some embodiments, the additional agent is a CEACAM inhibitor (for example, CEACAM-1, CEACAM-3 and / or CEACAM-5 inhibitor). In some embodiments, the CEACAM inhibitor is an anti-cCEACAM antibody molecule. Exemplary anti-cCEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, for example, a monoclonal antibody 34B1, 26H7 and 5F4; or a recombinant form thereof, as described in, for example, US 2004/0047858, US 7,132,255 and WO 99/052552. In some embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, for example, Zheng et al. PLoS One. (2011) 6 (6): e21146), or cross reactions with CEACAM-1 and CEACAM-5, as described in, for example, WO 2013/054331 and US 2014/0271618.
[00531] [00531] 4-1BB, also known as CD137, is a transmembrane glycoprotein belonging to the TNFR superfamily. 4-1BB receptors are present on activated T cells and B cells and monocytes. An exemplary anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and antineoplastic activities.
[00532] [00532] The tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), also known as OX40 and CD134, is another member of the TNFR superfamily. OX40 is not constitutively expressed in virgin T cells at rest and acts as a secondary co-stimulating immune checkpoint molecule. Exemplary anti-OX40 antibodies are MEDI6469 and MOXRO0916 (RG7888, Genen- tech).
[00533] [00533] In some embodiments, the additional agent includes a molecule that decreases the population of regulatory T cells (Treg). Methods that decrease the number of (e.g., deplete) Treg cells are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide and modulation of the gene function related to the glucocorticoid-induced TNFR family (GITR). GITR is a member of the TNFR superfamily that is regulated in activated T cells, which improves the immune system. Reducing the number of Treg cells in an individual before apheresis or before administration of modified cells, for example, CAR expression cells, can reduce the number of unwanted immune cells (eg, Tregs) in the tumor microenvironment and reduce the risk of relapse. In some embodiments, the additional agent includes a molecule that targets GITR and / or modulates GITR functions, such as a GITR agonist and / or a GITR antibody that depletes regulatory T cells (Tregs). In some embodiments, the additional agent includes cyclophosphamide. In some embodiments, the GITR-binding molecule and / or the molecule-modulating GITR function (e.g., GITR agonist and / or Treg-depleting GITR antibodies) is administered before the modified cells, e.g., expression cells CAR. For example, in some embodiments, the GITR agonist can be administered prior to apheresis of cells. In some embodiments, cyclophosphamide is administered to the individual prior to administration (for example, infusion or reinfusion) of the modified cells, for example, CAR expression cells or before the apheresis of the cells. In some embodiments, cyclophosphamide and an anti-GITR antibody are administered to the individual prior to administration (for example, infusion or reinfusion) of the modified cells, for example, CAR expression cells or before cell apheresis.
[00534] [00534] In some embodiments, the additional agent is a GITR agonist. Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (eg, divalent anti-GITR antibodies), such as, for example, a GITR fusion protein described in U.S. Patent American No. 6,111,090, European Patent No. 090505B 1, United States Patent 8,586,023, PCT Publications: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, for example, in the Northern Patent -American No. 7,025,962, European Patent No. 1947183B 1, United States Patent No. 7,812,135, United States Patent 8,388,967, United States Patent No. 8,591,886, European Patent No. EP 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WOZ2005 / 007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005 / 055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT publication No. WO99 / 20758, PCT publication No. WO2006 / 083289, PCT publication No. WO 2005/115451, United States Patent No. 7,618,632 and PCT Publication No. WO 2011/051726. An exemplary anti-GITR antibody is TRX518.
[00535] [00535] In some embodiments, the additional agent improves tumor infiltration or transmigration from administered cells, for example, CAR expression cells. For example, in some embodiments, the additional agent stimulates CD40, such as CDA40L, for example, recombinant human CD40L. Differentiation group 40 (CD40) is also a member of the TNFR superfamily. CD40 is a co-stimulating protein found in antigen-presenting cells and mediates a wide variety of immune and inflammatory responses. CD40 is also expressed in some malignant diseases, where it promotes proliferation. Exemplary anti-CD40 antibodies are da- cetuzumab (SGN-40), lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics) and CP-870,893. In some embodiments, the additional agent that improves tumor infiltration includes sunitnib tyrosine kinase inhibitors, heparanase and / or chemokine receptors, such as CCR2, CCR4 and CCR7.
[00536] [00536] In some embodiments, the additional agent includes drugs from thalidomide or its analogs and / or its derivatives, such as lenalide, pomalidomide or apremilast.
[00537] [00537] In some embodiments, the additional agent is a B cell inhibitor. In some embodiments, the additional agent is one or more B cell inhibitors selected from the CD10, CD19, CD20, CD22, CD34, CD123, CD79a inhibitors. , CD79b, CD179b, FLT-3 or ROR1 or a combination thereof. In some ways, the B cell inhibitor is an antibody (for example, a mono- or bispecific antibody) or its antigen-binding fragment. In some embodiments, the additional agent is a modified cell that expresses recombinant receptors that target B cell targets, for example, CD10, CD19, CD20, CD22, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3 or ROR1.
[00538] [00538] In some embodiments, the additional agent is a CD20 inhibitor, for example, an anti-CD20 antibody (for example, a mono or bispecific anti-CD20 antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab (also known as GA101 or RO5072759), veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab (also known as AME-133v or ocaratuzumab) (Genentech). See, for example, Lim et a /.
[00539] [00539] In some embodiments, the additional agent is a CD22 inhibitor, for example, an anti-CD22 antibody (for example, a mono or bispecific anti-CD22 antibody) or a fragment thereof. Exemplary anti-CD22 antibodies include epratuzumab and RFB4. In some embodiments, the CD22 inhibitor is a small molecule. In some embodiments, the antibody is a monospecific antibody, optionally conjugated to a second agent, such as a chemotherapeutic agent. For example, in some embodiments, the antibody is an anti-CD22-MMAE monoclonal antibody conjugate (for example, DCDT2980S). In some embodiments, the antibody is an scFv of an anti-CD22 antibody, for example, an scFv of the RFB4 antibody. In some embodiments, scFv is fused to all or to a fragment of exotoxin-A from Pseudomonas (for example, BL22). In some modes, scFv is fused to all or a fragment of (for example, a 38 kDa fragment of) Pseudomonas exotoxin-A (for example, pasoxotox moxetumomab). In some embodiments, the anti-CD22 antibody is a bispecific anti-CD19 / CD22 antibody, optionally conjugated to a toxin. For example, in some embodiments, the anti-CD22 antibody comprises a bispecific anti-CD19 / CD22 portion (for example, two scFv ligands, recognizing human CD19 and CD22) optionally linked to all or a portion of the diphtheria toxin (DT ), for example, first 389 amino acids of diphtheria toxin (DT), DT 390, for example, a ligand-directed toxin, such as DT2219ARL). In some embodiments, the bispecific portion (for example, anti-CD19 / anti-CD22) is linked to a toxin such as the deglycosylated ricin A chain (for example, Combo-tox).
[00540] [00540] In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the immunomodulatory agent is a cytokine or is an agent that induces increased expression of a cytokine in the tumor microenvironment. Cytokines have important functions related to T cell expansion, differentiation, survival and homeostasis. The cytokines that can be administered to the individual receiving the BCMA-binding receptors, cells and / or compositions provided herein include one or more than IL-2, IL-4, IL-7, IL-9, I1L-15, I1L-18 and 11-21. In some embodiments, the cytokine administered is IL-7, IL-15 or I1L-21, or a combination thereof. In some embodiments, administration of the cytokine to the individual who has a subideal response to the administration of the modified cells, for example, CAR expression cells improves the efficacy and / or antitumor activity of the administered cells, for example, expression cells CAR.
[00541] [00541] —By "cytokine" is meant a generic term for proteins released by a population of cells that act in another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocines and traditional polypeptide hormones. Included among cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin;
[00542] [00542] In some embodiments, the additional agent includes an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor (IL-15Ra) polypeptide or a combination thereof, for example, hetlL- 15 (Admune Therapeutics, LLC). hetlL-15 is a non-covalent heterodimeric complex of IL-15 and IL-15Ra. hetll-15 is described in, for example, United States Patent 8,124,084, United States Patent 2012/0177598, United States Patent 2009/0082299, United States Patent 2012/0141413 and United States Patent 2011/0081311 . In some embodiments, the immunomodulating agent may contain one or more cytokines. For example, interleukin may include leukocyte interleukin injection (Multikine), which is a combination of natural cytokines. In some modalities
[00543] [00543] In some embodiments, the additional agent is an agent that improves or neutralizes one or more toxicities or side effects associated with cell therapy. In some embodiments, the additional agent is selected from a steroid (eg, corticosteroid), a TNFα inhibitor and an I | L-6 inhibitor. An example of a TNFα inhibitor is an anti-TNFα antibody molecule, such as infliximabbe, adalimumab, certolizumab pegol and golimumab. Another example of a TNFα inhibitor is a fusion protein such as tanncept. Small molecule TNFα inhibitors include, but are not limited to, xanthine derivatives (eg, pesticide) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule, such as tocilizumab, sarilumab, elsilimomab, CNTO 328, ALD518 / BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX- 109, FE301 and FM101. In some embodiments, the anti-IL-6 antibody molecule is tocilizumab. In some embodiments, the additional agent is an inhibitor of IL-1R, such as anakinra.
[00544] [00544] In some embodiments, the additional agent is a modulator of adenosine levels and / or a component of the adenosine pathway. Adenosine can function as an immunomodulatory agent in the body. For example, adenosine and some adenosine analogs that selectively activate adenosine receptor subtypes decrease the production of inflammatory oxidative product neutrophils (Cronstein et al., Ann. NY Acad. Sci. 451: 291, 1985 ; Roberts et al., Biochem. J, 227: 669, 1985; Schrier et a., J. Inmunol. 137: 3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76, 1987). In some cases, the concentration of extracellular adenosine or adenosine analogues may increase in specific environments, for example, tumor microenvironment (TME). In some cases, signaling
[00545] [00545] —Inhibition or reduction of extracellular adenosine or adenosine receptor due to an extracellular adenosine inhibitor (as an agent that prevents the formation of, degrades, makes inactive and / or decreases extracellular adenosine) and / or an adenosine receptor inhibitor (such as an adenosine receptor antagonist) can improve the immune response, such as macrophages, neutrophils, granulocytes, dendritic cells, T-cell and / or B-mediated response. CAMP-dependent intracellular pathway mediated by Gs protein and inhibitors of intracellular pathways mediated by Gi protein activated by the adenosine receptor, can also increase acute and chronic inflammation.
[00546] [00546] In some modalities, the additional agent is an antagonist
[00547] [00547] An antagonist is any substance that tends to cancel the action of another, such as an agent that binds to a cellular receptor without causing a biological response. In some embodiments, the antagonist is a chemical compound that is an antagonist to an adenosine receptor, such as the A2a, A2b or A3 receptor. In some modalities, the antagonist is a peptide, or peptidomimetic, that binds to the adenosine receptor, but does not trigger an intracellular pathway dependent on the Gi protein. Exemplary antagonists are described in United States Patent 5,565,566; 5,545,627, 5,981,524; 5,861,405;
[00548] [00548] In some modalities, the additional agent is an antagonist
[00549] [00549] In some embodiments, the antagonist is an antisense molecule, nucleic acid inhibiting molecule (eg small inhibitory RNA (siRNA)) or catalytic nucleic acid molecule (eg a ribozyme) that specifically binds to the mMRNA that encodes an adenosine receptor. In some embodiments, the antisense molecule, nucleic acid inhibiting molecule or catalytic nucleic acid molecule binds to nucleic acids encoding A2a, A2b or A3. In some embodiments, an antisense molecule, nucleic acid inhibiting molecule or catalytic nucleic acid directs the biochemical pathways downstream of the adenosine receptor. For example, the antisense molecule or catalytic nucleic acid can inhibit an
[00550] [00550] In some embodiments, the additional agent that inhibits extracellular adenosine includes agents that make extracellular adenosine non-functional (or diminish that function), such as a substance that modifies the structure of adenosine to inhibit adenosine's ability to signal through adenosine receptors. In some embodiments, the additional agent is an extracellular adenosine generating or degrading enzyme, a modified form thereof or a modulator thereof. For example, in some modalities, the additional agent is an enzyme (for example, adenosine deaminase) or another catalytic molecule that selectively binds and destroys adenosine, abolishing or significantly decreasing the capacity of the adenosine formed endogenously to signal through adenosine receptors and end inflammation.
[00551] [00551] In some embodiments, the additional agent is an adenosine deaminase (ADA) or a modified form thereof, for example, recombinant ADA and / or ADA modified by polyethylene glycol (ADA-PEG), which can inhibit the accumulation extracellular adenosine in the local tissue. ADA-PEG was used to treat patients with ADA SCID (Hershfield (1995) Hum Mutat. 5: 107). In some modalities, an agent that inhibits extracellular adenosine includes agents that prevent or decrease the formation of extracellular adenosine and / or prevent or decrease the accumulation of extracellular adenosine, substantially abolishing or decreasing the immunosuppressive effects of adenosine. In some embodiments, the additional agent specifically inhibits enzymes and proteins involved in regulating the synthesis and / or secretion of pro-inflammatory molecules, including modulators of nuclear transcription factors. Suppression of adenosine receptor expression or expression of the Gs or Gi protein-dependent intracellular pathway or the CAMP-dependent intracellular pathway can result in an increase / enhancement of the immune response.
[00552] [00552] In some embodiments, the additional agent can target ectoenzymes that generate or produce extracellular adenosine. In some embodiments, the additional agent targets the CD39 and CD73 ectoenzymes, which work in tandem to generate extracellular adenosine. CD39 (also referred to as ectonucleoside triphosphate diphosphohydrolase) converts extracellular ATP (or ADP) into 5'AMP. Subsequently, CD73 (also referred to as 5'nucleotidase) converts 5'AMP to adenosine. CD39 activity is reversible by the actions of NDP kinase and adenylate kinase, while CD73 activity is irreversible. CD39 and CD73 are expressed in tumor stromal cells, including endothelial cells and Tregs, and also in many cancer cells. For example, the expression of CD39 and CD73 in endothelial cells is increased under the hypoxic conditions of the tumor microenvironment. Tumor hypoxia can result from inadequate blood supply and the disorganized vasculature of the tumor, impairing oxygen supply (Carroll and Ashcroft (2005), Expert. Rev. Mol. Med. 7 (6): 1-16). Hypoxia also inhibits adenylate kinase (AK), which converts adenosine to AMP, leading to a very high concentration of extracellular adenosine. Thus, adenosine is released in high concentrations in response to hypoxia, which is a condition that occurs frequently in the tumor microenvironment (TME), in or around solid tumors. In some embodiments, the additional agent is one or more anti-CD39 antibodies or their antigen binding fragment, anti-CD73 antibody or their antigen binding fragment, for example, MEDI9447 or TY / 23, aB- diphosphate methylene-adenosine (ADP), ARL 67156, POM-3, IPH52 (see, for example, Al-
[00553] [00553] In some embodiments, the additional agent is an inhibitor of hypoxia-inducible factor 1 alpha (HIF-10) signaling. Exemplary HIF-1a inhibitors include digoxin, acriflavin, sirtuin-7 and gandespib.
[00554] [00554] In some embodiments, the additional agent includes a protein tyrosine phosphatase inhibitor, for example, a protein tyrosine phosphatase inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, for example, an SHP-1 inhibitor described herein, such as, for example, sodium stiboglycolate. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor, for example, an SHP-2 inhibitor described herein.
[00555] [00555] In some embodiments, the additional agent is a kinase inhibitor. Kinase inhibitors, such as a CDKA4 kinase inhibitor, a BTK kinase inhibitor, an MNK kinase inhibitor or a DGK kinase inhibitor, can regulate the constitutively active survival pathways that exist in tumor cells and / or modulate the immune cell function. In some embodiments, the kinase inhibitor is a Bruton tyrosine kinase (BTK) inhibitor, for example, ibrutinib. In some embodiments, the kinase inhibitor is a phosphatidylinositol-4,5-bisphosphate 3-kinase (PISK) inhibitor. In some embodiments, the kinase inhibitor is a CDKA4 inhibitor, for example, a CDKA4 / 6 inhibitor. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as, for example, rapamycin, a rapamycin analogue, OSI-027. The mMTOR inhibitor can be, for example, an mMTORC1 inhibitor and / or an MTORCβ2 inhibitor, for example, an mMTORC1 inhibitor and / or MTORC2 inhibitor. In some modalities, the initiation
[00556] [00556] In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGlI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFEM-A13. In some embodiments, the BTK inhibitor does not reduce or inhibit interleukin-2-inducible kinase (ITK) kinase activity and is selected from GDC-0834; RN-486; CGI-560; CGI1- 1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A'13.
[00557] [00557] In some embodiments, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (1 - [(3R) -3- [4-Amino-3- (4-phenoxyphenyl) - 1H-pyrazole [3, 4-d] pyrimidin-1-yl] piperidin-1-yl] prop-2-en-1-one; also known as PCl-32765). In some embodiments, the kinase inhibitor is a BTK inhibitor, for example, ibrutinib (PClI-32765), and ibrutinib is administered in a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (for example, 250 mg, 420 mg or 560 mg) daily for a period of time, for example, daily - for a 21-day cycle, or daily for a 28-day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered. In some embodiments, the BTK inhibitor is a BTK inhibitor described in International Application WO 2015/079417.
[00558] [00558] In some embodiments, the kinase inhibitor is a PI3K inhibitor. PI3K is central to the PISK / AKVmTOR pathway involved in cell cycle regulation and lymphoma survival. Exemplary PI3K inhibitor includes idelalisib (PI3K5 inhibitor). In some modalities, the additional agent is idelalisib and rituximab.
[00559] [00559] In some embodiments, the additional agent is an inhibitor of the rapamycin mammalian target (MTOR). In some modalities, the kinase inhibitor is an MTOR inhibitor selected from temsirolimus; ridaforolimus (also known as AP23573 and MK8669); everolimus (RADO01); rapamycin (AY22989); simapimod; AZD8055; PFO4691502; SF1126; and XL765. In some embodiments, the additional agent is an inhibitor of mitogen-activated protein kinase (MAPK), such as vemurafenib, dabrafenib and trametinib.
[00560] [00560] In some embodiments, the additional agent is an agent that regulates pro or anti-apoptotic proteins. In some embodiments, the additional agent includes an inhibitor of B 2 cell lymphoma (BCL-2) (for example, venetoclax, also referred to as ABT-199 or GDC-0199; or ABT-737). Venetoclax is a small molecule (4- (4 - ([2- (4-chlorophenyl) -4,4-dimethyl-1-cyclohexen-1-ylmethyl) -1-piperazinyl) -N - ((3-nitro - 4 - [(tetrahydro-2H-pyran-4-ylmethyl) amino] phenyl) sulfonyl) -2- (1 H-pyrrolo [2,3- b] pyridin-5-yloxy) benzamide) that inhibits protein antiapoptotic, BCL-2. Other agents that modulate the pro or anti-apoptotic protein include the BCL-2 inhibitor ABT-737, navitoclax (ABT-263); Mcl-1 siRNA or Mcl-1 N- (4-hydroxyphenyl) retinoid (4-HPR) inhibitor for maximum effectiveness. In some modalities, the additional agent provides pro-apoptotic stimuli, such as the apoptosis-inducing ligand related to the recombinant tumor necrosis factor (TRAIL), which can activate the apoptosis pathway by binding to TRAIL death receptors DR-4 and DR-5 on the tumor cell surface, or agonistic antibodies to TRAIL-R2.
[00561] [00561] In some embodiments, the additional agent includes an indoleamine 2,3-dioxigenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid, L-tryptophan, to quinurrenine.
[00562] [00562] In some embodiments, the additional agent includes a cytotoxic agent, for example, CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarrubicin, or mitoxantrone. In some embodiments, the additional agent includes a hypomethylating agent, for example, a DNA methyltransferase inhibitor, for example, azacytidine or decitabine.
[00563] [00563] In another embodiment, the additional therapy is transplantation, for example, an allogeneic stem cell transplantation.
[00564] [00564] In some modalities, the additional therapy is a lymphodepletion therapy. Lymphoplete chemotherapy is thought to improve the graft and activity of recombinant receptor expression cells, such as CAR T cells. In some embodiments, lympho-exhaustion chemotherapy can enhance tumor-specific T cells adopted to proliferate in vivo through homeostatic proliferation (Grossman 2004, Stachel 2004). In some modalities, chemotherapy can reduce or eliminate CD4 + CD25 + regulatory T cells, which can suppress the function of adoptively transferred T cells directed to the tumor (Turk 2004). In some embodiments, lymphatic exhaustion chemotherapy prior to adoptive T cell therapy may enhance the expression of stromal cell-derived factor 1 (SDF-1) in the bone marrow, enhancing the return of the modified T cells to the primary tumor site by binding of SDF-1 with CXCR-4 expressed on the surface of T cells (Pinthus 2004). In some modalities, lymphatic drainage chemotherapy can further reduce the individual's tumor burden and potentially decrease the risk and severity of CRS.
[00565] [00565] In some modalities, lymphodepletion is performed on an individual, for example, before the administration of modified cells, for example, CAR expression cells. In some modalities, lymphodepletion comprises the administration of one or more of melphalan, Cytoxan, cyclophosphamide and / or fludarabine. In some modalities, a lindodepletora chemotherapy is administered to the individual before, simultaneously or after the administration (for example, infusion) of modified cells, for example, CAR expression cells. In one example, lymphatic-exhaustion chemotherapy is administered to the individual prior to the administration of modified cells, for example, CAR expression cells. In some embodiments, lymphoplasty chemotherapy is administered 1 to 10 days before the administration of modified cells, such as 1, 2, 3, 4, 5, 6.7, 8, 92 or days before the administration of modified cells, or at least 2 days before, as at least 3, 4, 5, 6 or 7 days before, at the beginning of the administration of the modified cell. In some modalities, the individual receives a preconditioning agent no more than 7 days before, as no more than 6, 5, 4, 3 or 2 days before,
[00566] [00566] In some modalities, the lymphatic drainage chemotherapy comprises the administration of a lymphodeplective agent, such as cyclophosphamide, fludarabine or combinations thereof. In some embodiments, the individual is administered cyclophosphamide at a dose between or between about 20 mg / kg and 100 mg / kg of body weight of the individual, such as between or between about 40 mg / kg and 80 mg / kg. In some aspects, the individual is administered with around 60 mg / kg of cyclophosphamide. In some embodiments, cyclophosphamide is administered once daily for one or two days. In some modalities, where the lymphodeplective agent comprises cyclophosphamide, is the individual administered with cyclophosphamide at a dose between or between about 100 mg / m and 500 mg / m of the individual's body surface, such as between or between about 200 mg / m and 400 mg / m or 250 mg / m and 350 mg / m , inclusive. In some cases, is the individual administered about 300 mg / m cyclophosphamide. In some modalities, cyclophosphamide can be administered in a single dose or in a plurality of doses, such as those administered daily, every two days or every three days. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 2 to 4 days. In some cases, is the individual given about 300 mg / m of the individual's body surface, of cyclophosphamide, daily for 3 days, before the beginning of cell therapy.
[00567] [00567] In some modalities, where the lymphodeplective agent comprises fludarabine, does the individual receive fludarabine in a dose between or between about 1 mg / m and 100 mg / m of the individual's body surface area, such as between or between about 10 mg / m and 75 mg / m , 15 mg / m and 50 mg / m , 20 mg / m and 40 mg / m or 24 mg / m and 35 mg / m , inclusive. In some cases, is the individual given about 30 mg / m of fludarabine. In some modalities, fluorabine can be administered in a single dose or in a plurality of doses, such as those administered daily, every two days or every three days. In some modalities, fludarabine is administered daily, such as for 1-5 days, for example, for 2 to 4 days. In some cases, is the individual given about 30 mg / m body surface area of fludarabine, daily for 3 days, before the start of cell therapy.
[00568] [00568] In some embodiments, the lymphodeplective agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents can include cyclophosphamide at any dose or schedule, as described above, and fludarabine at any dose or schedule, as described above. For example, in some ways, is the individual given fludarabine at or about 30 mg / m of the individual's body surface area, daily, and cyclophosphamide at or about 300 mg / m of the individual's body surface, daily, for 3 days.
[00569] [00569] In some modalities, antiemetic therapy, except dexamethasone or other steroids, can be administered prior to lymphoeswashing chemotherapy. In some modalities, Mesna can be used for individuals with a history of hemorrhagic cystitis.
[00570] [00570] In some embodiments, the additional agent is an oncolytic virus. In some embodiments, oncolytic viruses are able to selectively replicate and trigger death or slow the growth of a cancer cell. In some cases, oncolytic viruses have no effect or minimal effect on non-cancer cells. An oncolytic virus includes, but is not limited to, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccine virus, oncolytic synbis virus, oncolytic flu virus or oncolytic RNA virus (for example example, oncolytic reovirus, Newcastle oncolytic disease virus (NDV), oncolytic measles virus or oncolytic vesicular stomatitis (VSV) virus.
[00571] [00571] Other combination therapies, treatments and / or exemplary agents include antiallergic, antiemetic, analgesic and adjuvant therapies. In some embodiments, the additional agent includes cytoprotective agents, such as neuroprotectors, free radical scavengers, cardioprotectors, anthracycline leakage neutralizers and nutrients.
[00572] [00572] In some embodiments, an antibody used as an additional agent is conjugated or otherwise linked to a therapeutic agent, for example, a chemotherapeutic agent (for example, Cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, antitumor antibiotic, tyrosine kinase inhibitor, alkylating agent, antimicotubule or antimitotic agent), antiallergic agent, antinusea (or antiemetic) agent, analgesic or cytoprotective agent described here. In some embodiments, the additional agent is an antibody-drug conjugate.
[00573] [00573] In some embodiments, the additional agent may modulate, inhibit or stimulate particular factors in the levels of DNA, RNA or protein, to enhance or reinforce the effectiveness of BCMA-binding receptors, cells and / or recombinant compositions provided here - From. In some embodiments, the additional agent can modulate factors at the nucleic acid level, for example, DNA or RNA, within the cells administered, for example, cells modified to express recombinant receptors, for example, CAR. In some embodiments, an inhibitory nucleic acid, for example, an inhibitory nucleic acid, for example, a dsRNA, for example, a siRNA or ShRNA, or a regularly grouped interspersed short palindromic repeat (CRISPR), an activator of transcription as an effector nuclease (TALEN), or a zinc linker endonuclease (ZFN), can be used to inhibit the expression of an inhibitory molecule in the modified cell, for example, CAR expression cell. In some embodiments, the inhibitor is an sSshRNA. In some modalities, the inhibitory molecule is inhibited within the modified cell, for example, CAR expression cell. In some embodiments, a nucleic acid molecule that encodes a dsRNA molecule that inhibits the expression of the molecule that modulates or regulates, for example, inhibits, the function of T cells is operationally linked to a promoter, for example, a promoter derived from HI or U6 so that the dsRNA molecule that inhibits the expression of the inhibitory molecule is expressed within the modified cell, for example, CAR expression cell. See, for example, Brummelkamp TR, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19: 497-
[00574] [00574] In some embodiments, the additional agent is capable of disrupting the gene encoding an inhibitory molecule, such as any immune checkpoint inhibitors described herein. In some embodiments, interruption occurs by deletion, for example, deletion of a gene, exon or entire region and / or substitution with an exogenous sequence and / or by mutation, for example, reading or reading phase mutation. incorrect sense, within the gene, typically within an exon of the gene. In some modalities, the rupture results in the
[00575] [00575] In some respects, disruption is accomplished by editing genes, such as using a DNA-binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the gene in a targeted region rupture. In some aspects, the protein or nucleic acid is coupled or complexed with a nuclease, as in a chimeric or fusion protein. For example, in some embodiments, disruption is performed using a fusion comprising a DNA targeting protein and a nuclease, such as a Zinc Link Nuclease (ZFN) or TAL effector nuclease (TALEN) or a guided nuclease by RNA, such as a regularly clustered interspaced short palindromic nucleic acid (CRISPR) -Cas system, such as the CRISPR-Cas9 system, specific for the gene being disrupted. In some modalities, methods of producing or generating genetically modified cells, for example, CAR expression cells, include introducing into a population of cell nucleic acid molecules that co-challenge a genetically modified antigen receptor (for example, CAR) and nucleic acid molecules that encode an agent that targets an inhibitory molecule that is a gene editing nuclease, such as a fusion of a DNA targeting protein and a nuclease like a ZFN or a TALEN, or an RNA-guided nuclease like the CRISPR-Cas9 system, specific for an inhibitory molecule.
[00576] [00576] Any of the additional agents described herein can be prepared and administered as combination therapy with the recombinant BCMA-binding receptor (e.g., chimeric antigen receptor) and / or modified cells that express said molecules. (for example, recombinant receptor) described herein, as in pharmaceutical compositions comprising one or more combination therapy agents and a pharmaceutically acceptable carrier, such as any described herein. In some modalities, the BCMA-binding recombinant receptor (for example, chimeric antigen receptor), modified cells that express said molecules (for example, recombinant receptor), plurality of modified cells that express said molecules ( for example, recombinant receptor) can be administered simultaneously, concurrently or sequentially, in any order with the additional agents, therapy or treatment, in which this administration provides therapeutically effective levels for each of the agents in the individual's body. In some embodiments, the additional agent may be co-administered with the BCMA-binding recombinant receptors, cells and / or compositions described herein, for example, as part of the same pharmaceutical composition or using the same delivery method. In some embodiments, the additional agent is administered simultaneously with the BCMA-binding recombinant receptors, cells and / or compositions described herein, however, in separate compositions. In some embodiments, the additional agent is an additional modified cell, for example, a cell modified to express a different recombinant receptor, and is administered in the same composition or in a separate composition. In some embodiments, the additional agent is incubated with the modified cell, for example, CAR expression cells, prior to administration of the cells.
[00577] [00577] In some examples, one or more additional agents are administered subsequently or prior to administration of the BCMA-binding recombinant receptors, cells and / or compositions described herein, separated by a selected period of time. In some instances, the time period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, | month, 2 months or 3 months. In some examples, the one or more additional agents are administered multiple times and / or the recombinant BCMA-binding receptors, cells and / or compositions described herein, are administered multiple times. For example, in some embodiments, the additional agent is administered prior to the BCMA-binding recombinant receptors, cells and / or compositions described herein, for example, two weeks, 12 days, 10 days, 8 days, one week, 6 days, days, 4 days, 3 days, 2 days or 1 day before administration. For example, in some embodiments, the additional agent is administered after the BCMA-binding recombinant receptors, cells and / or compositions described herein, for example, two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after administration.
[00578] [00578] The dose of the additional agent can be any therapeutically effective amount, for example, any amount of dose described herein, and the appropriate dosage of the additional agent can depend on the type of disease being treated, the type, dose and / or frequency of the recipient, cell and / or recombinant composition administered, the severity, and the course of the disease, if the recipient, cell and / or recombinant composition is administered for preventive or therapeutic purposes, prior therapy, clinical history of the patient and response to matching receiver, cell and / or composition, and the discretion of the attending physician. The recipient, cell and / or recombinant composition and / or the additional agent and / or therapy can be administered to the patient at the same time, repeated or administered over a series of treatments.
[00579] [00579] “Manufacture articles or kit containing the supplied recombinant receptors (for example, CARs), genetically modified cells and / or compositions comprising them are also provided. Manufacturing articles may include a container and a label or insert for packaging in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags of solution | V, etc. Containers can be formed from a variety of materials, such as glass or plastic. In some embodiments, the container has a sterile access door. Exemplary containers include bags of intravenous solution, flasks, including those with needle-piercing plugs for injection. The manufacturing article or kit may also include a packaging insert indicating that the compositions can be used to treat a particular condition, such as a condition described herein (for example, multiple myeloma). Alternatively, or in addition, the article of manufacture or kit may include still another or the same container comprising a pharmaceutically acceptable buffer. It may also include other materials, such as other buffers, thinners, filters, needles and / or syringes.
[00580] [00580] The label or insert of the packaging may indicate that the composition is used to treat the disease, disorder or condition of BCMA expression or associated with BCMA, in an individual. The label or insert of the packaging that is in or associated with the container, can indicate instructions for reconstitution and / or use of the formulation. The label or insert of the packaging may also indicate that the formulation is useful or intended for subcutaneous, intravenous or other modes of treatment or prevention of a disease, disorder or condition of expression of BCMA or associated with BCMA in an individual.
[00581] [00581] The container in some modalities has a composition that is by itself or combined with another composition effective to treat, prevent and / or diagnose the condition. The manufacturing article or kit may include (a) a first container with a composition contained therein (ie, first drug), in which the composition includes the antibody (for example, anti-BCMA antibody) or fragment thereof binding to recombinant antigen or receptor (for example, CAR); and (b) a second container with a composition contained therein (i.e., second medicine), in which the composition includes an additional agent, such as a cytotoxic or therapeutic agent, and whose article or kit further comprises instructions on the label or on the label. packaging insert to treat the individual with the second medication, in an effective amount. VII. DEFINITIONS
[00582] [00582] “When used here, reference to a" corresponding form "of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form as the antibody. For example, if an antibody is declared to have greater activity compared to the activity of the corresponding form of a first antibody, this means that a particular form, such as an scFv of this antibody, has greater activity compared to the form of scFv of the first antibody.
[00583] [00583] The term "Fc region" here is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxy terminal of the heavy chain. However, C-terminal lysine (Lys447) from the Fc region may or may not be present. Unless otherwise specified here, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
[00584] [00584] The terms "full-length antibody", "intact antibody" and "whole antibody" are used here interchangeably to refer to an antibody that has a structure substantially similar to a native antibody structure or that has chains that contain a region of Fc as defined herein.
[00585] [00585] "An" isolated "antibody is one that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to more than 95% or 99% purity, as determined by, for example, electrophoretic (eg, SDS-PAGE, isoelectric focus (IEF), capillary electrophoresis) or chromatographic (eg, exchange ion or reverse phase HPLC). For review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).
[00586] [00586] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, however, the nucleic acid molecule is present in an extrachromosomal form or in a chromosomal location different from its natural chromosomal location.
[00587] [00587] "Isolated nucleic acid encoding an anti-BCMA antibody" refers to one or more nucleic acid molecules that co-complicate and light and heavy chains of antibody (or fragments thereof), including such (such) molecule ( s) nucleic acid in a single vector or separate vectors, and such (such) nucleic acid molecule (s) present in one or more locations in a host cell.
[00588] [00588] The terms "host cell", "host cell lineage" and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acid has been introduced, including the progeny of such cells . Host cells include "transformants" and "transformed cells", which include the transformed primary cell, and the progeny derived from them without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to a mother cell, however, it may contain mutations. Mutant progeny that have the same biological function or activity as screened or selected in the originally transformed cell are included here.
[00589] [00589] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including antibodies and chains of antibodies and other peptides, for example, BCMA-binding ligands and peptides, can include amino acid residues, including natural and / or unnatural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation and the like. In some aspects, polypeptides may contain modifications in relation to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as by means of site-directed mutagenesis, or they may be accidental, such as by means of host mutations that produce proteins or errors due to PCR amplification.
[00590] [00590] “When used here," percentage (%) of amino acid sequence identity "and" percentage of identity "and" identity
[00591] [00591] An amino acid substitution may include replacing an amino acid in a polypeptide with another amino acid. Amino acid substitutions can be introduced into a binding molecule, for example, antibody, of interest and the products screened for a desired activity, for example, retained / improved antigen binding or reduced immunogenicity.
[00592] [00592] Amino acids can generally be grouped according to the following common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lle; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence the orientation of the chain: Gly,
[00593] [00593] Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.
[00594] [00594] The term "vector", when used here, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it was introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operationally linked. Such vectors are referred to here as "expression vectors".
[00595] [00595] The term "packaging insert" is used to refer to the instructions normally included in commercial packages of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.
[00596] [00596] “When used here, the singular forms" um "," uma "and" alo "include plural referents, unless the context clearly indicates otherwise. For example, "one" or "one" means "at least one" or "one or more". It is understood that aspects, modalities and variations described herein include "comprising", "consisting" and / or "consisting essentially of" aspects, modalities and variations.
[00597] [00597] - Along with this description, several aspects of the object claimed are presented in an interval format. It should be understood that the description in the interval format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the subject matter claimed. Therefore, the description of a range should be considered as having specifically described all possible sub-ranges, as well as
[00598] [00598] The term "about", when used here, refers to the usual error range for the respective value easily known by the person skilled in this technical field. The reference to "about" a value or parameter here includes (and describes) modalities that target this value or parameter per se. For example, the description for "about X" includes the description for "X".
[00599] [00599] “When used here, a" composition "refers to any mixture of two or more products, substances or compounds, including cells. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination of them.
[00600] [00600] “When used here, a statement that a cell or cell population is" positive "for a specific marker refers to the detectable presence in the cell of a specific marker or in the cell of a particular marker, typically a marker of surface. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and by detecting said antibody. , in which the staining is detectable by flow cytometry at a level substantially above the detected staining by performing the same procedure with an isotype-matched control under otherwise identical conditions and / or at a substantially similar level to the cell being knows to be positive for the marker and / or at a substantially higher level than for a cell known to be negative for the marker.
[00601] [00601] “When used here, a statement that a cell or population of cells is" negative "for a specific marker refers to the absence of a substantial detectable presence in or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of expression of the surface as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and by detecting said antibody, where staining is not detected by flow cytometry at a level substantially above the detected staining by performing the same procedure with an isotype-matched control under identical conditions, and / or at a level substantially less than that for a known cell known be positive for the marker and / or at a substantially similar level compared to that for a known cell known to be negative for the marker.
[00602] [00602] Unless otherwise defined, all terms of the technique, notations and other technical or scientific terms or terminology used here have the same meaning as is commonly understood by someone skilled in the technique to which the claimed subject matter belongs. . In some cases, terms with commonly understood meanings are defined here for clarity and / or for immediate reference, and the inclusion of such definitions here should not necessarily be interpreted.
[00603] [00603] All publications, including patent documents, scientific articles and databases, referred to in this application, are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication were incorporated individually by reference. . If a definition mentioned here is contrary or otherwise inconsistent with a definition mentioned in patents, orders, published orders and other publications that are incorporated herein by reference, the definition mentioned here takes precedence over the definition that is incorporated here by reference.
[00604] [00604] The section titles used here are for organizational purposes only and should not be interpreted to limit the subject matter described. VIII. EXEMPLARY MODALITIES
[00605] [00605] Among the modalities provided here are:
[00606] [00606] 1.0 The polynucleotide encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, where after expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (MRNA), of the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90% or 95% homogeneity of RNA.
[00607] [00607] 2. The polynucleotide of embodiment 1, wherein the spacer is derived from an immunoglobulin.
[00608] [00608] 3. The polynucleotide of Embodiment 1 or Embodiment 2, wherein the spacer comprises a sequence of a hinge region, a region of Ch42 and Cr3.
[00609] [00609] 4. The polynucleotide of any of the modalities 1-3, wherein the encoded spacer is or comprises (i) the sequence mentioned in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity for SEQ ID NO: 649; or (iii) a contiguous portion of (i) or (ii) with at least 125 amino acids in length.
[00610] [00610] 5. The polynucleotide of any of modalities 1-4, wherein the nucleic acid encoding the spacer comprises at least one modified binding donor and / or binding receptor site, said modified binding donor and / or receptor site comprising one or more nucleotide modifications corresponding to a reference binding donor site and / or reference binding receptor site contained in the sequence mentioned in SEQ ID NO: 621.
[00611] [00611] 6. The polynucleotide of embodiment 5, wherein the one or more nucleotide modifications comprise an insertion, deletion, substitution or combinations thereof.
[00612] [00612] 7.The polynucleotide of embodiment 5 or embodiment 6, wherein the reference binding receptor sites and / or reference binding donors are canonical, non-canonical or cryptic binding sites.
[00613] [00613] 8. The polynucleotide of any of the 5-7 modalities, in which: the reference binding donor site (s) and / or reference binding receptor (s) have (have) ) a binding site prediction score of at least or about 0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 , 0.99 or 1.0; and / or the referral link donor site (s) and / or referral link receiver (s) is / are expected to be involved in a liaison event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[00614] [00614] 9.0 polynucleotide of any of the 5-8 modalities, wherein: the donor site of the reference link comprises the sequence aatctaagtacggac (SEQ ID NO: 705), teaactagtacgatag (SEQ ID NO: 706), acaattagtaaggca (SEQ ID NO: 707) and / or accacaggtgatatac (SEQ ID NO: 708); and / or the reference binding receptor site comprises the sequence aagtttctttetatatteccaggcetgacegtggataaatete (SEQ ID NO: 742) and / or gggcaacdgtattctettgcagtateatacacgaagecetage (SEQ ID NO: 743).
[00615] [00615] 10. The polynucleotide of any of the 5-8 modalities, in which: the reference binding donor site (s) and / or reference binding receptor (s) have (have) ) a binding site prediction score of at least or about 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99 or 1.0; and / or the referral donor site (s) and / or referral link receiver (s) is (are) expected to be involved ) in a liaison event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[00616] [00616] 11.The polynucleotide of any of modalities 5-8 and 10, wherein: the donor site of the reference link comprises the tcaactagtacgtag sequence (SEQ ID NO: 706); and / or the reference binding receptor site comprises the sequence aagtttctttetatattecaggetgacegtggataaatete (SEQ ID NO: 742).
[00617] [00617] 12. The polynucleotide of any of the 5-11 modalities, wherein at least one of the one or more nucleotide modifications is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues from the junction of the reference binding receptor site and / or reference binding donor site.
[00618] [00618] 13. The polynucleotide of any of the modalities 5-12, in which the one or more nucleotide modifications are silent and / or result in a degenerate codon in comparison to SEQ ID NO: 621 and / or do not alter the sequence of amino acids of the coded spacer.
[00619] [00619] 14. The polynucleotide of any of modalities 5-9 and 12-13, wherein: the modified donor binding site is mentioned in agtttaaatacggac (SEQ ID NO: 661), teaactagtatatag (SEQ ID NO: 662) , accatctccaaggcce (SEQ ID NO: 663) and / or gcececaggtttacac (SEQ ID NO: 664); and / or the modified binding receptor site is mentioned in ca- gtttcttectatatagtagactcaccgtggataaatcaa (SEQ ID NO: 672), gggcaacg- tatteagctgcagcegtgatgcacgaggcectac (SEQ ID NO: 673) and / or cagecctta-.
[00620] [00620] 15. The polynucleotide of any of modalities 5-14, in which the modified donor binding site is mentioned in tcaac-tagtatatag (SEQ ID NO: 662) and / or the modified recipient site is mentioned in cagtttcttcctatatagtagactcaccegtggataaatcaa ( SEQ ID NO: 672) / or cgccettgtectectigtecegetectectatigecggacet (SEQ ID NO: 766).
[00621] [00621] 16. The polynucleotide of any of embodiments 1-15, wherein the spacer is encoded by a nucleotide sequence mentioned in SEQ ID NO: 622 or a portion thereof.
[00622] [00622] 17. A polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide comprises the nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is either comprised of the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d)
[00623] [00623] 18. Polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide comprises the nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid consists or essentially consists of the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[00624] [00624] 19. The polynucleotide of Embodiment 17 or Embodiment 18, wherein after expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (MRNA), of the polynucleotide, exhibits at least 70%, 75 %, 80%, 85%, 90% or 95% homogeneity of the RNA.
[00625] [00625] 20. The polynucleotide of any of modalities 1-19, in which, after expression in a cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide exhibits reduced heterogeneity compared to the heterogeneity of the mRNA transcribed from a reference polynucleotide, said reference polynucleotide which encodes the same amino acid sequence as the polynucleotide, in which the reference polynucleotide differs by the presence of one or more donor and / or binding sites one or more receptor binding sites on the nucleic acid encoding the spacer and / or comprises one or more nucleotide modifications compared to the polynucleotide.
[00626] [00626] 21.0 The polynucleotide of embodiment 20, wherein the RNA heterogeneity is reduced by more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more .
[00627] [00627] 22.0 The polynucleotide of Embodiment 20 or Embodiment 21, wherein the RNA transcribed, optionally RNA
[00628] [00628] 23.0 The polynucleotide of any of the modalities 1-22, in which RNA homogeneity and / or heterogeneity is / are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation , fractionation of field flow or liquid chromatography.
[00629] [00629] 24.0 polynucleotide of any of the modalities 1-23, in which the polynucleotide is codon optimized.
[00630] [00630] 25.0 The polynucleotide of any of the modalities 1-24, in which the antigen is associated with the disease or condition or expressed in cells of the environment of an injury associated with the disease or condition.
[00631] [00631] 26.0 The polynucleotide of any of the modalities 1-25, in which the disease or condition is a cancer.
[00632] [00632] 27.0 The polynucleotide of any of the modalities 1-26, in which the disease or condition is a myeloma, leukemia or lymphoma.
[00633] [00633] 28. The polynucleotide of any of modalities 1-27, in which the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2 / neu (thyrosin erbB2 receptor kinase), LI-CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2) , epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vlll, folate-binding protein (FBP), FOCRL5, FOCRH5, fetal acetylcholine receptor, GD2 , GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insertion domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule, (LI-CAM ), Melanoma-associated antigen (MAGE) -A1,
[00634] [00634] 29. The polynucleotide of embodiment 28, wherein the antigen is B cell maturation antigen (BCMA).
[00635] [00635] 30. The polynucleotide of any of modalities 1-29, wherein the antigen binding domain is an antibody fragment comprising a heavy chain variable region (Vn) and a light chain variable (V.) .
[00636] [00636] 31. Embodiment polynucleotide 30, wherein: the Vx region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609 , 617, 772-774, or 814-832; and / or the V region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116-
[00637] [00637] 32.The polynucleotide of Embodiment 30 or Embodiment 31, wherein: the Vx region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% sequence identity for the Va region amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252 , 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522,609 or 617; and / or the V region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the Vi region amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326 , 327, 534, 535, 536, 537, 538, 610 or 618.
[00638] [00638] 33. The polynucleotide of Embodiment 30 or Embodiment 31, wherein: the V4 region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vn region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and / or the V. region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 116-127, 257-267, 326 , 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
[00639] [00639] 34. The polynucleotide of any of the modalities 30-33, wherein: the Vx region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the selected Vn region amino acid sequence of any of the SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522,609 or 617; and / or the V. region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 116, 117, 118, 120, 121 , 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
[00640] [00640] 35. The polynucleotide of any of the modalities 30- 34, in which: the Vu region is or comprises (a) a heavy chain 1 complementarity determining region (CDR-H1) that comprises the sequence amino acid selected from any of SEQ ID NOs: 1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) comprising the selected amino acid sequence from any of SEQ ID NOs: 4-6, 145- 148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the selected amino acid sequence from any of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517,595, 606, 613; and / or the V. region is or comprises (a) a light chain 1 complementarity determining region (CDR-L1) comprising the selected amino acid sequence from any of SEQ ID NOs: 26-36 , 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determination region 2 (CDR-L2) comprising the selected amino acid sequence from any of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409 , 411-414, 590,602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the selected amino acid sequence from any of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429- 433,591 or 603.
[00641] [00641] 36.0 The polynucleotide of any of the modalities 30-35, in which: the Vx region is or comprises (a) a heavy chain 1 complementarity determining region (CDR-H1) that comprises the sequence of amino acids selected from any of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604,611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) comprising the selected amino acid sequence from any of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain 3 complementarity determining region (CDR-H3) comprising the selected amino acid sequence from any of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517,595, 606 or 613; and / or the V region is or comprises (a) a light chain 1 complementarity determining region (CDR-L1) comprising the selected amino acid sequence from any of SEQ ID NOs: 26, 27 , 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain 2 complementarity determining region (CDR-L2) comprising the selected amino acid sequence from any of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182 , 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determination region 3 (CDR-L3) comprising the selected amino acid sequence from any of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194,
[00642] [00642] 37.0 The polynucleotide of any of the modalities 30-36, in which the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 1.4, and 7, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 10, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 3, 6, and 11, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 140, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 141, 145, and 150, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 142, 146, and 151, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 152, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 143, 147, and 153, respectively
[00643] [00643] 38.0 The polynucleotide of any of the modalities 30-37, in which the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 1.4, and 7, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 10, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively;
[00644] [00644] 39. The polynucleotide of any of the modalities 30-38, wherein the Vx region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531 , 533, 609, 617, 772-774, or 814-832.
[00645] [00645] 40. The polynucleotide of any of the modalities 30-39, wherein the Vx region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115,248, 252, 253, 254 , 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617.
[00646] [00646] 41.The polynucleotide of any of the 30-40 modalities, wherein: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 593, 594, and 595 , respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively.
[00647] [00647] 42.0 polynucleotide of any of the modalities 30-41, wherein the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617.
[00648] [00648] 43. The polynucleotide of any of the modalities
[00649] [00649] 44. The polynucleotide of any of the modalities 30-43, in which the Vi region. comprises a CDR-L1, CDR-L2 and CDR-L3 selected from: a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 26, 37, and 47, respectively - you; a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 27, 38, and 48, respectively; a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 28, 39, and 49, respectively; a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 30, 39, and 51, respectively; a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 31, 41, and 52, respectively; a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 33, 43, and 55, respectively; a CDR-L1, CDR-L2 and CDR-L3 comprising the
[00650] [00650] 45.The polynucleotide of any of the modalities 30-44, wherein the Vi region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550 , 552-557, 610, 618, 775-777, or 833-849.
[00651] [00651] 46.0 The polynucleotide of any of the modalities 30-45, wherein the Vi region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
[00652] [00652] 47. The polynucleotide of any of the modalities 30-46, wherein: region V. comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; or the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 614, 615, and 603, respectively.
[00653] [00653] 48.The polynucleotide of any of the 30-47 modalities, wherein a V region is or comprises the amino acid sequence mentioned in SEQ ID NO: 618.
[00654] [00654] 49.0 The polynucleotide of any of the modalities 30-48, in which: the V "region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence which has at least 90% identity for SEQ ID NO: 110 and 116, respectively; the V "region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively, or a sequence amino acids that have at least 90% identity to SEQ ID NO: 111 and 117, respectively; the V region "and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 118, respectively;
[00655] [00655] 50. A polynucleotide of any of the modalities 30- 49, in which: the V "region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or a sequence of amino acids that have at least 90% identity to SEQ ID NO: 110 and 116, respectively; the V "region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively, or a amino acid sequence that has at least 90% identity to SEQ ID NO: 111 and 117, respectively;
[00656] [00656] 51.0 The polynucleotide of any of the 30-50 modalities, wherein the fragment comprises a scFv.
[00657] [00657] 52.0 The polynucleotide of any of the modalities 30-51, when the Vu region, and the V. region are joined by a flexible ligand.
[00658] [00658] 53.0 The polynucleotide of embodiment 52, wherein the scFv comprises a linker comprising the amino acid sequence GGGGSGGGGSGCGGGGS (SEQ ID NO: 361).
[00659] [00659] 54.0 The polynucleotide of any of the 30-53 modalities, in which the Vx region is amino terminal to the V region.
[00660] [00660] 55.0 The polynucleotide of any of the 30-54 modalities, wherein the antigen-binding domain comprises the amino acid sequence selected from any of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585 or 769-771 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % sequence identity for the selected amino acid sequence of any of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585 or 769-
[00661] [00661] 56.0 The polynucleotide of any of the 30-55 modalities, wherein the antigen binding domain comprises the selected amino acid sequence from any of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the selected amino acid sequence of any of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 .
[00662] [00662] 57.0 The polynucleotide of any of the 30-56 modalities, wherein the nucleic acid encoding the antigen binding domain comprises (a) the nucleotide sequence mentioned in any of SEQ ID NOS: 330-352, 647 , 648, 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity to any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b).
[00663] [00663] 58.0 The polynucleotide of any of embodiments 30-57, wherein the nucleic acid encoding the antigen binding domain comprises (a) the nucleotide sequence mentioned in any of SEQ ID NOS: 352, 647, 648 , 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity to any of SEQ ID NOS: 352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b).
[00664] [00664] 59. The polynucleotide of any of the 30-57 modalities, wherein the nucleic acid encoding the antigen-binding domain is codon-optimized.
[00665] [00665] 60. The polynucleotide of any of embodiments 30-57, wherein the nucleic acid encoding the antigen binding domain comprises the nucleotide sequence mentioned in any of SEQ ID NO: 440, 460, 715, 717 or 719 .
[00666] 61. The polynucleotide of any of the 30-60 embodiments, wherein the nucleic acid encoding the antigen binding domain comprises the nucleotide sequence mentioned in SEQ ID NO: 460.
[00667] [00667] 62.0 The polynucleotide of any of the modalities 30-53, in which the Vu region is carboxy-terminal for the V region.
[00668] [00668] 63.The polynucleotide of any of the modalities 51-53 and 62, wherein the scFv comprises the amino acid sequence mentioned in SEQ ID NOs: 328 or 586, or an amino acid sequence that has at least 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the amino acid sequence mentioned in SEQ ID NO: 328 or 586.
[00669] [00669] 64. The polynucleotide of any of modalities 1-59, wherein the intracellular signaling region comprises an activation cytoplasmic signaling domain.
[00670] [00670] 65. The polynucleotide of embodiment 60, wherein the activation cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a component of the T cell receptor (TCR) and / or comprises an immunoreceptor tyrosine-based activation motif (ITAM).
[00671] [00671] 66. The polynucleotide of Embodiment 64 or Embodiment 65, wherein the activation cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a zeta chain of a CD3-zeta chain (CD36) or a functional variant or signaling portion thereof.
[00672] [00672] 67.0 The polynucleotide of any of the 64-66 modalities, in which the cytoplasmic activation domain is human or is derived from a human protein.
[00673] [00673] 68.The polynucleotide of any of the 64-67 modalities, wherein the cytoplasmic activation domain is or comprises the sequence mentioned in SEQ ID NO: 628 or a sequence of amino acids that is at least 90% sequence identity for SEQ ID NO: 628.
[00674] [00674] 69.0 The polynucleotide of any of the 64-68 modalities, wherein the nucleic acid encoding the cytoplasmic activation domain is or comprises the sequence mentioned in SEQ ID NO: 627 or is a codon-optimized sequence and / or a degenerate sequence.
[00675] [00675] 70. The polynucleotide of any of the 64-69 modalities, wherein the nucleic acid encoding the cytoplasmic signaling domain of activation is or comprises the sequence mentioned in SEQ ID NO: 652.
[00676] [00676] 71.The polynucleotide of any of the 64-70 modalities, wherein the intracellular signaling region further comprises a co-stimulating signaling region.
[00677] [00677] 72. The polynucleotide of embodiment 71, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof.
[00678] [00678] 73.0 The polynucleotide of Embodiment 71 or Embodiment 72, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4- 1BB or an ICOS or a signaling portion thereof .
[00679] [00679] 74.0 The polynucleotide of any of the modalities 71-73, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
[00680] [00680] 75.The polynucleotide of any of the modalities 71-74, in which the co-stimulatory signaling region is human or is derived from a human protein.
[00681] [00681] 76.0 The polynucleotide of any of the modalities 71-75, wherein the co-stimulatory signal region is or comprises the sequence mentioned in SEQ ID NO: 626 or an amino acid sequence that exhibits at least 90% sequence identity for the sequence mentioned in SEQ ID NO: 626.
[00682] [00682] 77.0 The polynucleotide of any of the modalities 71-76, wherein the nucleic acid encoding the co-stimulatory region is or comprises the sequence mentioned in SEQ ID NO: 625 or is a codon-optimized and / or degenerated sequence of the month - ma.
[00683] [00683] 78.0 The polynucleotide of any of the modalities 71-77, wherein the nucleic acid encoding the co-stimulating signal region comprises the sequence mentioned in SEQ ID NO: 681.
[00684] [00684] 79.0 The polynucleotide of any of the modalities 71-78, wherein the co-stimulatory signaling region is between the transmembrane domain, and the intracellular signaling region.
[00685] [00685] 80. The polynucleotide of any of modalities 1-79, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD4, CD28 or CD8.
[00686] [00686] 81. The polynucleotide of embodiment 80, wherein the transmembrane domain is or comprises a trans28 membrane domain derived from a CD28.
[00687] [00687] 82.0 The polynucleotide of any of modalities 1-81, wherein the transmembrane domain is human or is derived from a human protein.
[00688] [00688] 83.The polynucleotide of any of the modalities 1-82, wherein the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 624 or an amino acid sequence that exhibits at least 90% sequence identity to SEQ ID NO: 624.
[00689] [00689] 84. The polynucleotide of any of modalities 1-83, wherein the nucleic acid encoding the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 623 or is a codon-optimized and / or degenerate sequence of the same.
[00690] [00690] 85. The polynucleotide of embodiment 35a, wherein the nucleic acid encoding the transmembrane domain comprises the sequence mentioned in SEQ ID NO: 688.
[00691] [00691] 86. The polynucleotide of any of modalities 1-85, in which the encoded chimeric antigen receptor comprises its N to C terminal in order: the antigen-binding domain, the spacer, the transmembrane domain, and the intracellular signaling domain.
[00692] [00692] 87. The polynucleotide of any of the modalities 1-86, wherein the polynucleotide further encodes a truncated receptor.
[00693] [00693] 88. A chimeric antigen receptor encoded by the polynucleotide of any of modalities 1-87.
[00694] [00694] 89. A chimeric antigen receptor comprising: (a) an extracellular antigen-binding domain that specifically recognizes the B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.
[00695] [00695] 90. The chimeric antigen receptor of embodiment 89, wherein the spacer is derived from an immunoglobulin.
[00696] [00696] 91. The chimeric antigen receptor of embodiment 89 or embodiment 90, wherein the spacer comprises a sequence of a hinge region, a Ch2 and Ch3 region.
[00697] [00697] 92. The chimeric antigen receptor of embodiment 91, in which one of more than the joint, Cx2 and Cnr3 is derived wholly or partially from IGG4 or IgG2, optionally human I9G4 or human I9gG2.
[00698] [00698] 93. The chimeric antigen receptor of embodiment 90 or embodiment 91, wherein the joint, Ch2 and CH3 is derived from I9G4.
[00699] [00699] 94. The chimeric antigen receptor of embodiment 90 or embodiment 91, wherein one or more of the joints of Cr2 and Ch3 are chimeric and comprise the sequence derived from IgG4 and IgG2.
[00700] [00700] 95.The chimeric antigen receptor of embodiment 94, wherein the spacer comprises a chimeric IgG4 / 2 joint or one of modified IgG4 that comprises at least one amino acid substitution compared to human IgG4, a chimeric CH> 2 region of I9G2 / 4 and a Cr3 of I9G4.
[00701] [00701] 96. The chimeric antigen receptor of any of the 89-92 and 94-95 modalities, wherein the spacer is or comprises (i)
[00702] [00702] 97. The chimeric antigen receptor of any of the 89-92 and 94-96 modalities, wherein the encoded spacer is or comprises the sequence mentioned in SEQ ID NO: 649.
[00703] [00703] 98. A chimeric antigen receptor comprising: (a) an extracellular antigen-binding domain that specifically recognizes the B cell maturation antigen (BCMA); (b) a spacer mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[00704] [00704] 99 The chimeric antigen receptor of any of the 89-98 modalities, wherein the antigen-binding domain is an antibody fragment comprising a variable heavy chain (Vn) and variable light chain (V.) region .
[00705] [00705] 100. The chimeric antigen receptor of embodiment 99, wherein: the Vx region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% sequence identity for the V4 region amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256,324, 325, 518-531 , 533, 609 or 617; and / or the V region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the Vi region amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-
[00706] [00706] 101.The chimeric antigen receptor of embodiment 99 or embodiment 100, wherein: the Vx region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Va region amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522,609 or 617; and / or the V region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity for the V region amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
[00707] [00707] 102. The chimeric antigen receptor of embodiment 99 or embodiment 100, wherein: the Vx region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence Vx region selected from any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and / or the V. region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 116-127, 257-267, 326 , 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
[00708] [00708] 103. The chimeric antigen receptor of any of the 99-102 modalities, wherein: the V4 region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vx region selected from any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521,
[00709] [00709] 104. The chimeric antigen receptor of any of the 99-103 modalities, wherein: the Vx region is or comprises (a) a heavy chain 1 complementarity determining region (CDR-H1) that comprises comprises the selected amino acid sequence of any of SEQ ID NOs: 1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) comprising the selected amino acid sequence from any of SEQ ID NOs: 4-6, 145- 148, 290, 291, 296, 297,372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the selected amino acid sequence from any of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517,595, 606, 613; and / or the V. region is or comprises (a) a light chain 1 complementarity determining region (CDR-L1) comprising the selected amino acid sequence from any of SEQ ID NOs: 26-36 , 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determination region 2 (CDR-L2) comprising the selected amino acid sequence from any of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409 , 411-414, 590,602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the selected amino acid sequence from any of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429- 433,591 or 603.
[00710] [00710] 105. The chimeric antigen receptor of any of the 99-104 modalities, wherein: the Vx region is or comprises (a) a heavy chain 1 complementarity determining region (CDR-H1) that comprises comprises the selected amino acid sequence of any of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604,611; and / or (b) a heavy chain 2 complementarity determining region (CDR-H2) comprising the selected amino acid sequence from any of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain 3 complementarity determining region (CDR-H3) comprising the selected amino acid sequence from any of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517,595, 606 or 613; and / or the V region is or comprises (a) a light chain 1 complementarity determining region (CDR-L1) comprising the selected amino acid sequence from any of SEQ ID NOs: 26, 27 , 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain 2 complementarity determining region (CDR-L2) comprising the selected amino acid sequence from any of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182 , 183, 304, 305, 399, 400, 401, 402, 590, 602,608 or 615; and (c) a light chain complementarity determination region 3 (CDR-L3) comprising the selected amino acid sequence from any of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418,421, 591, or 603.
[00711] [00711] 106. The chimeric antigen receptor of any of the 99-105 modalities, in which a Vx region comprises a CDR-H1, CDR-H2 and CDR-H3, selected from:
[00712] [00712] 107. The chimeric antigen receptor of any of the 99-106 modalities, in which a Vx region comprises a CDR-H1, CDR-H2 and CDR-H3, selected from: a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 1.4, and 7, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 8, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 9, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 2, 5, and 10, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 141, 145, and 149, respectively; a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 143, 147, and 153, respectively
[00713] [00713] 108. The chimeric antigen receptor of any of the 99-107 modalities, wherein a Vx region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-
[00714] [00714] 109 The chimeric antigen receptor of any of the 99-108 modalities, wherein a Vx region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 110, 111, 112, 113, 115,248, 252 , 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617.
[00715] [00715] 110. The chimeric antigen receptor of any of the 99-109 modalities wherein: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 593, 594, and 595, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 611, 612, and 613, respectively;
[00716] [00716] 111. The chimeric antigen receptor of any of the 99-110 modalities, wherein a Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617.
[00717] [00717] 112. The chimeric antigen receptor of any of the 99-111 modalities, in which a V. region comprises a
[00718] [00718] 114. The chimeric antigen receptor of any of the 99-113 modalities, wherein the V. region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 116-127, 257-267, 326 , 327, 534-550, 552-557, 610, 618, 775-777, or 833-
[00719] [00719] 115. The chimeric antigen receptor of any of the 99-114 modalities, wherein the V. region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 116, 117, 118, 120, 121 , 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
[00720] [00720] 116. The chimeric antigen receptor of any of the 99-115 modalities, wherein: region V. comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; or the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 614, 615, and 603, respectively.
[00721] [00721] 117. The chimeric antigen receptor of any of the 99-116 modalities, wherein the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 618.
[00722] [00722] 118. The chimeric antigen receptor of any of the 99-117 modalities, wherein: the V "region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 116, respectively; the V "region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively , or an amino acid sequence that has at least 90% identity to SEQ ID NO: 111 and 117, respectively; the V region "and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 118, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 118, respectively;
[00723] [00723] 119. A chimeric antigen receptor of any of the 99-118 modalities, wherein: the V "region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 110 and 116, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NO: 110 and 116, respectively; the V "region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 111 and 117, respectively , or an amino acid sequence that has at least 90% identity to SEQ ID NO: 111 and 117, respectively;
[00724] [00724] 120. The chimeric antigen receptor of any of the 99-119 modalities, wherein the fragment comprises an scFv.
[00725] [00725] 121. The chimeric antigen receptor of any of the 99-120 modalities, when the VH region and the Vi region are joined by a flexible ligand.
[00726] [00726] 122.The chimeric antigen receptor of embodiment 121, wherein the scFv comprises a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 361).
[00727] [00727] 123. The chimeric antigen receptor of any of the 99-122 modalities, wherein the VH region is amino-terminal to the V ..
[00728] [00728] 124. The chimeric antigen receptor of any of the 99-123 modalities, wherein the antigen-binding domain comprises the selected amino acid sequence of any of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558- 576, 578- 583, 585 or 769-771 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity for the selected amino acid sequence from any of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585 or 769-771.
[00729] [00729] 125. The chimeric antigen receptor of any of the 99-124 modalities, wherein the antigen-binding domain comprises the selected amino acid sequence of any of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273- 278, 329, 442, 478, 558-563 or 585 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% sequence identity for the selected amino acid sequence from any of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329,442, 478 , 558-563 or 585.
[00730] [00730] 126. The chimeric antigen receptor of any of the 99-122 modalities, in which the Va region is a carboxy-terminal to the region
[00731] [00731] 127. The chimeric antigen receptor of any of the 99-122 and 126 modalities, wherein the scFv comprises the amino acid sequence mentioned in SEQ ID NOs: 328 or 586, or an amino acid sequence that is at least 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the amino acid sequence mentioned in SEQ ID NO: 328 or 586.
[00732] [00732] 128. The chimeric antigen receptor of any of the 89-127 modalities, wherein the intracellular signaling region comprises a cytoplasmic signaling activation domain.
[00733] [00733] 129.0 The chimeric antigen receptor of embodiment 128, in which the activation cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a component of the T cell receptor (TCR ) and / or comprises an activation motif based on tyrosine immunoreceptor (ITAM).
[00734] [00734] 130. The chimeric antigen receptor of embodiment 128 or embodiment 129, wherein the activation cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a zeta chain of a chain of CD3-zeta (CD37) or a functional variant or signaling portion thereof.
[00735] [00735] 131. The chimeric antigen receptor of any of the 128-130 modalities, wherein the cytoplasmic activation domain is human or is derived from a human protein.
[00736] [00736] 132. The chimeric antigen receptor of any of the 128-131 embodiments, wherein the cytoplasmic activation domain is or comprises the sequence mentioned in SEQ ID NO: 628 or an amino acid sequence that has at least 90% identity of SEQ ID NO: 628.
[00737] [00737] 133. The chimeric antigen receptor of any of the 128-132 modalities, wherein the intracellular signaling region further comprises a co-stimulating signaling region.
[00738] [00738] 134.The chimeric antigen receptor of embodiment 133, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof.
[00739] [00739] 135.The chimeric antigen receptor of embodiment 133 or embodiment 134, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a portion signaling.
[00740] [00740] 136. The chimeric antigen receptor of any of the modalities 133-135, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
[00741] [00741] 137. The chimeric antigen receptor of any of the 133-136 modalities, wherein the co-stimulatory signaling region is human or is derived from a human protein.
[00742] [00742] 138. The chimeric antigen receptor of any of the modalities 133-137, wherein the co-stimulatory signaling region is or comprises the sequence mentioned in SEQ ID NO: 626 or an amino acid sequence that exhibits at least 90% identity. - sequence quality for the sequence mentioned in SEQ ID NO:
[00743] [00743] 139. The chimeric antigen receptor of any of the 133-139 modalities, wherein the co-stimulatory signaling region is between the transmembrane domain, and the intracellular signaling region.
[00744] [00744] 140. The chimeric antigen receptor of any of the 89-139 modalities, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD4, CD28 or
[00745] [00745] 141. The chimeric antigen receptor of embodiment 140, wherein the transmembrane domain is or comprises a transmembrane domain derived from a CD28.
[00746] [00746] 142. The chimeric antigen receptor of any of the 89-141 modalities, wherein the transmembrane domain is human or is derived from a human protein.
[00747] [00747] 143. The chimeric antigen receptor of any of the 89-142 modalities, wherein the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 624 or an amino acid sequence that exhibits at least 90 % sequence identity for SEQ ID NO: 624.
[00748] [00748] 144. The chimeric antigen receptor of any of the 89-143 modalities, wherein the encoded chimeric antigen receptor comprises its N to C terminal in order: the antigen binding domain, the spacer, the transmembrane domain, and the intracellular signaling domain.
[00749] [00749] 145. A modified cell, comprising the polynucleotide of any of modalities 1-87 and 173-180.
[00750] [00750] 146. A modified cell, comprising the chimeric antigen receptor of any of the modalities 88-144 and 181.
[00751] [00751] 147. The modified cell of embodiment 145 or embodiment 146, wherein the cell is an immune cell.
[00752] [00752] 148. The modified cell of embodiment 147, wherein the immune cell is a primary cell obtained from an individual.
[00753] [00753] 149. The modified cell of embodiment 147 or embodiment 148, wherein the immune cell is an NK cell or a T cell.
[00754] [00754] 150. The modified cell of any of the 147-149 modalities, wherein the immune cell is a T cell, and the T cell is a CD4 + and / or CD8 + T cell.
[00755] [00755] 151. The cell modified from any of the 145-150 modalities, wherein the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (MRNA), which exhibits at least 70%, 75%, 80% , 85%, 90%, or 95% RNA homogeneity.
[00756] [00756] 152. The cell modified from any of the 145-151 modalities, wherein the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (MRNA), which exhibits reduced heterogeneity compared to MRNA heterogeneity transcribed in a cell encoding a reference chimeric antigen receptor, said reference chimeric antigen receptor that comprises the same amino acid sequence as the chimeric antigen receptor, however, encoded by a polynucleotide sequence different which comprises one or more nucleotide differences in the polynucleotide encoding the CARs and / or in which the reference chimeric antigen receptor is encoded by a polynucleotide comprising one or more donor binding sites and / or one or more binding receptor sites in the nucleic acid encoding the spacer.
[00757] [00757] 153. The modified cell of embodiment 152, wherein the RNA heterogeneity is reduced by more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more.
[00758] [00758] 154. The modified cell of Embodiment 152 or Embodiment 153, wherein the cell encoding the reference CAR comprises the transcribed RNA encoding the reference CAR, optionally the messenger RNA (mMRNA), which exhibits more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity.
[00759] [00759] 155. The cell modified from any of the modalities
[00760] [00760] 156. The modified cell of any of the 145-155 modalities, in which, out of a plurality of modified cells, less than or less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2% or 1% of cells in plurality comprise a chimeric antigen receptor that exhibits tonic signaling and / or antigen-independent activity or signaling.
[00761] [00761] 157. A composition comprising the polynucleotide of any of modalities 1-87 and 173-179, the chimeric antigen receptor of any of modalities 88-144 and 180, or the modified cell of any of modalities 144- 156.
[00762] [00762] 158. The composition of embodiment 157, which further comprises a pharmaceutically acceptable excipient.
[00763] [00763] 159.The composition of Embodiment 157 or Embodiment 158, which is sterile.
[00764] [00764] 160. A method of treatment, which comprises administering modified cells of any of the 144-156 modalities or the composition of any of the 157-159 modalities to an individual with a disease or disorder.
[00765] [00765] 161. The method of embodiment 160, in which the disease or disorder is associated with the expression of the B cell maturation antigen (BCMA).
[00766] [00766] 162. The method of any embodiment 160 or embodiment 161, wherein the disease or disorder associated with BCMA is a B cell-related disorder.
[00767] [00767] 163. The method of any of the modalities 160-162, in which the disease or disorder associated with BCMA is an autoimmune disease or disorder.
[00768] [00768] 164. The method of embodiment 163, wherein the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, hip-associated vasculitis, idiopathic thrombocytopenic purpura (ITP), purplish thrombotic thrombocytopenia (TTP), autoimmune thrombocytopenia, Chagas disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, polyps, nephropathy, nephropathy - IgM neuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, Reynaud's syndrome, antiphospholipid syndrome, Goodpasture's disease, Kawasaki's disease, autoimmune hemolytic anemia, severe myasthenia or progressive glomerulonephritis.
[00769] [00769] 165. The method of any of the 160-164 modalities, in which the disease or disorder associated with BCMA is a cancer.
[00770] [00770] 166.The embodiment method 165, in which cancer is a BCMA-expressing cancer.
[00771] [00771] 167.The method of Embodiment 165 or 166, wherein the cancer is a B-cell malignancy.
[00772] [00772] 168. The method of any of the modalities165-167, in which the cancer is a lymphoma, a leukemia or a malignancy of plasma cells.
[00773] [00773] 169. Embodiment 168 method, wherein the cancer is a lymphoma, and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, lymphoma follicular, non-cleaved small cell lymphoma, mucosal lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma so, angiocentric pulmonary B cell lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmicocytic lymphoma) or lining cell lymphoma (MCL).
[00774] [00774] 170. The embodiment method 168, in which the cancer is leukemia, and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL).
[00775] [00775] 171. The method of embodiment 168, in which cancer is a malignancy of plasma cells, and the malignancy of plasma cells is multiple myeloma (MM) or plasmacytoma.
[00776] [00776] 172. The method of any of the modalities 165-168 and 171, in which the cancer is multiple myeloma (MM).
[00777] [00777] 173. The polynucleotide of any of modalities 1-87, wherein the antigen binding domain and / or the encoded chimeric antigen receptor exhibits preferential binding to and / or exhibits greater binding affinity to the membrane-bound BCMA in comparison to soluble BCMA.
[00778] [00778] 174. The polynucleotide of any of modalities 1-87 and 173, wherein the polynucleotide comprises the sequence mentioned in any of SEQ ID NOS: 751-762 or a sequence that exhibits at least 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity for the sequence mentioned in any of the SEQs ID NOS: 751- 762 and maintains the function of binding to BCMA and keeps the RNA heterogeneity reduced.
[00779] [00779] 175. The polynucleotide of any of modalities 1-87, 173 and 174, wherein one or more of the joint, CH2 and Ch3, is derived wholly or partially from IgG4 or IgG2, optionally human IgG4 or human IgG2.
[00780] [00780] 176.The polynucleotide of any of modalities 1-87, 173-175, in which the joint, Ch2 and Cr3, is derived from IgG4.
[00781] [00781] 177. The polynucleotide of any of modalities 1-87 and 173-176, in which one or more of the joint, Cx2 and Ch3, is chimeric and comprises the sequence derived from IgG4 and IgG2.
[00782] 178. The polynucleotide of embodiment 177, wherein the spacer comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint comprising at least one amino acid substitution compared to human IgG4, a chimeric Ch62 region of I9G2 / 4 and one of Cr3 of IgG4.
[00783] [00783] 179. The polynucleotide of any of modalities 1-87 and 173-178, wherein the encoded spacer is or comprises the sequence mentioned in SEQ ID NO: 649.
[00784] [00784] 180. The chimeric antigen receptor of any of the 88-144 modalities, wherein the antigen-binding domain and or the chimeric antigen receptor exhibit preferential binding to, and / or exhibit greater binding affinity to the BCMA bound to membrane compared to soluble BCMA.
[00785] [00785] 181.The chimeric antigen receptor of any of 181. The chimeric antigen receptor of any of the 88- 144 modalities, wherein the antigen binding domain and or the chimeric antigen receptor, or an indicative measure the function or activity of the chimeric antigen receptor encoded after exposure to cells that express surface BCMA, is not reduced or blocked or is not substantially reduced or blocked in the presence of a soluble or spillable form of BCMA.
[00786] [00786] 182. The chimeric antigen receptor of embodiment 181, wherein the concentration or amount of the soluble or spillable form of BCMA corresponds to a concentration or amount present in the serum or blood or plasma of the individual or a patient with multiple myeloma or, on average, in a patient population for the disease or disorder, or in a concentration or amount of soluble or spillable BCMA in which the binding or measurement is reduced
[00787] [00787] 183. Method for determining the heterogeneity of a nucleic acid transcribed from a transgene, the method comprising: a) amplifying a transcribed nucleic acid using at least a 5 'and 3' primer pair, where at least one pair comprises a 5th primer that is complementary to a nucleic acid sequence within the 5 '(5' RTU) untranslated region of the transcribed nucleic acid and a 3 'primer that is complementary to a sequence of nucleic acid within the 3 '(3' UTR) untranslated region of the transcribed nucleic acid to generate one or more amplified products; and b) detecting the amplified products, in which the presence of two or more amplified products from at least one 5 'and 3' primer pair indicates heterogeneity in the amplified products.
[00788] [00788] 184. The method of embodiment 183, in which the differences detected in b) are different lengths from the amplified transcripts.
[00789] [00789] 185. The method of embodiment 183, in which the differences in b) are differences in the chromatographic profiles of the amplified transcripts.
[00790] [00790] 186. The method of any of the modalities 183-185, in which the differences in the amplified products are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation or chromatography.
[00791] [00791] 187. The method of any of the modalities 183-186, in which the 5 'primer is specific to the transcribed sequence of the region
[00792] 188. The method of any of the modalities 183-187, wherein the transcribed nucleic acid is amplified using a 3 'primer specific to a sequence within the amino acid coding sequence of the polynucleotide and / or the untranslated region 3 'of the transcribed pre-mRNA.
[00793] [00793] 189. The method of any of the modalities 183-188, wherein the 3 'primer is specific to the polyadenylation sequence or enhancer region of the 3' untranslated region of the transcribed pre-mRNA.
[00794] [00794] 190. The method of any of the modalities 183-189, in which step a) is performed by a single amplification reaction, using a single pair of 5 'and 3' primers comprising a 5 'which is complementary to a nucleic acid sequence within the 5' (5 'UTR) untranslated region of the transcribed nucleic acid and a 3' primer which is complementary to a nucleic acid sequence within the 3 'untranslated region ( 3 'RTU).
[00795] [00795] 191. The method of any of the modalities 183-190, in which step a) is carried out by parallel or subsequent amplification reactions using a first pair of initiators in 5 and 3, a second pair of initiators in 5 'e 3' and optionally 5 'and 3' primer pairs, where: the first 5 'and 3' primer pair contains a 5 'primer which is complementary to a nucleic acid sequence within the 5 'Transcribed nucleic acid RTU and a 3' primer which is complementary to a nucleic acid sequence within the 3 'transcribed nucleic acid RTU; the second 5 'and 3' primer pair contains a 5 'primer whose sequence is complementary to a portion of the translated nucleic acid transcription sequence and a 3' primer whose sequence is complementary to a nucleic acid sequence within the 3 'transcription RTU; and the optionally additional 5 'and 3' primer pairs each contain sequences complementary to the sequences within the translated region of the transcription.
[00796] [00796] 192. The method of embodiment 191, in which parallel or subsequent amplification reactions amplify the overlapping portions of the transcript.
[00797] [00797] 193. The method of any of the modalities 183-192, in which the amplified products are predicted to be about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 chi - lobases, 4.5 kilobases, 5 kilobases, 5.5 kilobases, 6 kilobases, 7 kilobases or 8 kilobases in length.
[00798] [00798] 194. The method of any of the modalities 183-193, wherein a transcribed nucleic acid that is detected as having heterogeneity is identified as a candidate for transgene for the removal of one or more binding sites.
[00799] [00799] 195. The embodiment method 194, wherein the transgene candidate's transcribed nucleic acid exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40 %, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity after expression in a cell.
[00800] [00800] 196. A method for reducing the heterogeneity of an expressed transgene transcription, the method comprising: a) identifying a transgene candidate for the removal of binding sites according to the method of embodiment 194 or embodiment 195; b) identify one or more potential donor and / or binding receptors sites; and c) modifying the nucleic acid sequence at or near one or more binding donor sites identified in b), thereby generating a modified polynucleotide.
[00801] [00801] 197. The method of embodiment 196, further comprising: d) evaluating the transgenic candidacy for the removal of binding sites, as in step a).
[00802] [00802] 198. The method of embodiment 197, further comprising e) repeating steps b) -d) until the heterogeneity of the transcription in step d) is reduced compared to the heterogeneity of the transcription, as determined in step a).
[00803] [00803] 199. The method of any of the 196-198 modalities, in which the one or more potential donor and / or binding receptor sites exhibit a score of about or at least about 0.7 , 0.75, 0.8, 0.85, 0.9, 0.95 or 1.0 of a link event or probability of a link event.
[00804] [00804] 200.The method of any of the 196-199 modalities, wherein the binding donor and binding receptor sites are independently identified.
[00805] [00805] 201.The method of any of the 196-200 modalities, wherein the recipient site (s) and / or binding donor (s) is / are recipient site (s) or canonical (s), non-canonical (s) and / or cryptic (s) binding donor (s).
[00806] [00806] 202.The method of any of the 196-201 modalities, wherein the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor.
[00807] 203. The method of embodiment 202, wherein the CAR polypeptide comprises an antigen-binding domain comprising an antibody fragment, optionally a single chain antibody (scFv) fragment, comprising a variable weight chain (Va) and a variable light chain (V1), a spacer region, a transmembrane region and an intracellular signaling region.
[00808] [00808] 204. The method of embodiment 202 or embodiment 203, wherein the modified polynucleotide is not modified within the coding sequence for the antigen binding domain of the encoded CAR polypeptide.
[00809] [00809] 205.The method of any of the 196-204 modalities, wherein the encoded amino acid sequence of the transgene is unchanged after modification of the polynucleotide.
[00810] [00810] 206.The method of any of the 196-205 modalities, wherein the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80%, 85%, 90%, 95% or 95% homogeneity after expression of the unmodified polynucleotide in a cell.
[00811] [00811] 207. The method of any of the modalities 183-206, in which the cell is a human cell.
[00812] [00812] 208.The method of any of the modalities 183-207, in which the cell is a T cell. IX. EXAMPLES
[00813] [00813] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: Generation and evaluation of anti-BCMA antibodies (Vu chain only)
[00814] [00814] Exemplary anti-BCMA antibodies containing a heavy chain variable region (Vr) that specifically binds to BCMA, even in the absence of a light chain variable region (V.), were generated and evaluated. A. Library Selection and Antibody Generation
[00815] [00815] Several BCMA-binding Vu regions were generated through a series of selection steps performed on members of a Vx library of human normal donor antibody labeled with
[00816] [00816] Exemplary Vx clones that exhibit specific binding to cell lines expressing BCMA, but not to BCMA negative control cells were sequenced and purified for further characterization. The clones were purified and titrated, and their binding affinities (ECso) to nBCMA / HEK293 cells were measured using an assay based on flow cytometry with fluorochrome-conjugated anti-His antibody. Table E1 lists sequences from the heavy chain complementarity determination region 3 (CDR-H3) of exemplary clones, containing framework regions derived from human V43 and their respective binding affinities (EC50) observed in this study.
[00817] [00817] Exemplary anti-BCMA antibodies, formatted as fragments of single chain antibodies (scFvs), were identified and evaluated for binding to BCMA. A. Library Selection and Generation of scFv Antibodies
[00818] [00818] Exemplary anti-BCMA scFv antibodies were generated through various selections, performed on libraries of normal human donor antibodies encoded by dsDNA, displayed in a cell-free system. In one method, members of the Vx region library enriched from a first screening cycle in the method described in Example 1 were matched by rearrangement with members of a normal human donor Vi library to generate a scFv library, in VH- (G4S) 3-V1 format. The resulting scFv libraries were enriched in subsequent rounds of selection for specific binding to BCMA expression HEK293 cells compared to the original HEK293 cells.
[00819] [00819] In another method, the de novo selection was performed by screening a human scFv library derived from a normal donor for specific binding of BCMA to hBCMA-Fc in the presence or absence of competitive elution with a ref scFv antibody. - mouse anti-BCMA ratio (BCMA-C1, Vi-Vn scFv antibody, SEQ ID NO: 328; or BCMA-C 2, Vu-V. scFv antibody, SEQ ID NO: 329). After at least 2 selection cycles, the scFv ligands were recovered.
[00820] [00820] —Specific binding of resulting scFv clones to BCMA expression HEK293 cells, compared to control cells that do not express BCMA, was assessed by flow cytometry with crude cell lysate translated in vitro or with produced supernatant by bacteria. Certain scFv clones that exhibited binding preference for BCMA were also analyzed.
[00821] [00821] The selected scFv clones were sequenced using the forward and reverse primers and purified for another characterization. Table E2 lists the sequence identifiers (SEQ ID NO) corresponding to the amino acid (aa) and nucleotide (nt) sequences of the scFv and amino acid sequences of the corresponding variable regions of the heavy chain (VH) or light chain (V. ), CDRs and framework regions (FRs). With regard to the BCMA-22 clone, the first residue of the CDR3 light chain (a cysteine), which was inherited from the germline structure region, was replaced by a serine to generate an additional scFv, designated BCMA-23. Table E2 also mentions the sequence of exemplary mouse anti-BCMA reference antibodies used as controls and in competition studies, as described in the subsequent Examples.
[00822] [00822] “Exemplary clones were purified and titrated and their binding affinities (EC50) for hBMCA were tested: BCMA-1, BCMA-2, BCMA-3, BCMAH4, BOCMA-5, BCMA-6, BOCMA-7 , BOCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-14, BCMA-15, BCMA-16, BCMA-17, BCMA-18, BCMA -19, BCMA -20, BCMA-21, BCMA-22, BCMA-23, BOMA-24, BCMA-25, BCMA-26, BCMA-27, BCMA-28 and BCMA-29. Other anti-BCMA scFv antibodies were also evaluated (see, Table E3), such as scFvs containing Vi and Vi sequences of antibodies described in WO2016090327 and scFvs containing Vx and V. sequences of BCMA antibodies described in WO2010104949.
[00823] [00823] —Pollucleotides encoding exemplary chimeric antigen receptors (CARs), each containing a binding domain to the anti-human BCF scFv antigen, have been generated. Among the human anti-BCMA scFvs were those described in Example 2. Also among the CARs generated were CARs containing scFvs containing VH and VL sequences of antibodies described in WO2016090327. Anti-BCMA CARs containing scFvs with BCH antibody VH and VL sequences described in WOZ2010104949 were also generated. In some cases of scFv, Vu was amino-terminal to V. and, in some cases, V. was amino-terminal to Vu. The scFv regions in generated CARs are mentioned in Table E4.
[00824] [00824] Specifically, exemplary polynucleotide CAR constructs contained nucleic acid encoding a human IgG-kappa signal sequence (SEQ ID NO: 619, encoding SEQ ID NO: 620), a human anti-BCMA scFv ( SEQ ID NOS: 128 - 130, 132, 133, 136, 137, 269, 273-277, 442, 478 and 558-563), a spacer (such as a spacer containing Chn2-CH3 modified IgG4 joint (SEQ ID NO: 621, encoding SEQ ID NO: 649) (whose spacer may in some cases be referred to as "LS") or, in some cases, a shorter spacer (which may be referred to as "SS") ,
[00825] [00825] A polynucleotide encoding another CAR construct was also generated containing nucleic acid encoding a human IgG-kappa signal sequence (SEQ ID NO: 619, encoding SEQ ID NO: 620), an anti-BCMA scFv of mouse (SEQ ID NO: 328 (BCMA-C1; V.-VH), 329 (BCMA-C2; Vu-V.), 585 (BCMA-C1; V4-VL) or 586 (BCMAC-2; V.- VH)), a spacer (SEQ ID NO: 621, encoding SEQ ID NO: 649), a human CD28 transmembrane domain, an intracellular cosine signaling sequence derived from human 4-1BB and an intracellular signaling domain derived from CD3-zeta.
[00826] [00826] The cDNA clones encoding these CARs were linked to a downstream ribosomal hop element (such as the T2A coding sequence SEQ ID NO: 686 or 687, encoding SEQ ID NO: 654) followed by a truncated receiver coding sequence
[00827] [00827] To generate T cells expressing anti-BCMA CAR, T cells were isolated by immunoaffinity-based enrichment from leukapheresis samples from human donor individuals. The isolated T cells were activated and transduced with lentiviral vectors containing the respective polynucleotides that encode the anti-BCMA CAR. On transduction and expansion, CD4 + and CD8 + T cells were stained with an antibody specific for the truncated receptor and with a recombinant human BCMA labeled with fluorose, and analyzed by flow cytometry, confirming cell transduction and expression anti-BCMA CARs. Example 4: Assessment of potential RNA heterogeneity and modification
[00828] [00828] RNA from cells transduced with exemplary anti-BCMA CARs, as described in Example 3, was analyzed for heterogeneity by agarose gel electrophoresis, after polymerase chain reaction with reverse transcriptase (RT-PCR ) using promoter-specific initiators, and the WPRE downstream in the SUTR and 3'UTR of the exemplary CAR transcripts. Several bands were observed for several anti-BCMA CAR constructs containing an exemplary spacer, including a modified IgG Cr2-CH3 hinge region (BCMA-LS CAR) (FIG. 1A), indicating RNA heterogeneity. Less RNA heterogeneity was observed for exemplary CARs containing a shorter spacer, such as one that includes a portion of an extracellular region of human CD28 (see, for example, BCMA-52-SS CAR).
[00829] [00829] In the nucleotide sequences encoding several BCMA-LS CARs, they were evaluated for potential binding sites and modified conservatively, including the removal of potential predicted binding sites. Sequences prior to modification
[00830] [00830] The modified sequences were constructed and tested for heterogeneity of RNA, as described above. Electrophoresis confirmed the reduction in RNA heterogeneity. Analysis of BCMA-CAR constructs before and after elimination of the binding site demonstrated reduced RNA heterogeneity (FIG. 1B). Exemplary O / SSE CAR constructions were generated containing modifications of the long spacer region, for example, BCMA-23-LS-O / SSE CAR, BCMA-25-LS-O / SSE CAR, BCMA-26- CAR LS-O / SSE, BCMA-52-LS-O / SSE CAR and BCMA-55-LS-O / SSE CAR. Example 5: Evaluation of CAR expression and function in primary T cells
[00831] [00831] Lentiviral constructs containing polynucleotides encoding anti-BCMA CAR with starting and optimized sequences, respectively, as described in Example 3, were transduced into T cells and the transduced cells were analyzed for transduction (based on expression of a substitute marker) and CAR expression based on binding to recombinant BCMA-Fc fusion protein by flow cytometry. A higher percentage of CD4 + and CD8 + T cells transduced using the optimized sequences, BCMA-52-LS-O / SSE CAR and BCMA-55-LS-O / SSE CAR, expressed the anti-BCMA CAR in surface, compared to those transduced to express the same corresponding CAR through the polynucleotide that has the starting sequence (not SSE). Representative data is mentioned in FIG 2 and Table E5 below. Anti-BCMA CAR
[00832] [00832] Various volumes of viral preparations containing lignor vectors encoding CAR constructions, BCMA-23-LS CAR, BCMA 26-LS CAR, BCMA 55-LS CAR and BCMA 55-LS-O / CAR SSE, were used to transduce 500,000 donor-derived primary human T cells and the transduction efficiency was compared. The percentage of T cell transduction was increased after transduction by optimized sequences (FIG. 3, circles) compared to the starting sequences (FIG. 3, triangles). Example 6: Characterization of BCMA-52 and BCMA-55 scFvs A. Tissue Staining by Immunohistochemistry
[00833] [00833] Cells and tissues expressing varying levels of BCMA were assessed by immunohistochemistry for binding of exemplary anti-BCMA antibodies. Binding domains (scFvs) of exemplary human BCMA-targeted CARs, which were fused with a peptide from the mouse IGG1 Fc region, were assessed for cell and tissue binding by immunohistochemistry. B. Evaluation of Binding Kinetics
[00834] [00834] A CAR with a BCMA-55 derived scFv binding domain, a modified IgG-derived ChH2-CH3 joint spacer, a CD28 transmembrane domain and 41BB and CD3zeta endodomain were expressed in a T cell lineage Jurkat. CAR binding kinetics to recombinant human BCMA-hFc (rhB-CMA hFc) were evaluated using a kinetics exclusion assay. The affinity of binding of an Fc fusion protein containing the scFv portion of the CAR (scFv-Fc) to the recombinant human BCMA fusion protein was also assessed using a Biacore-based assay. In these studies, the Kp for binding by fusion of CAR and scFv-Fc, respectively, was observed to be approximately 1 nM and 10 nM.
[00835] [00835] In another experiment, Jurkat cells were transduced with a polynucleotide that encodes a CAR with a BCMA-55-derived scFv binding domain and were grown at a density of - 2 x 10º. The cells were harvested and centrifuged at 1500g for 15 minutes at 4ºC. The cell pellet was washed and the cells were resuspended and serially diluted in biotinylated rhBCMA hFc at nM or 1 nM (also referred to in this assay as the constant ignition partner (CBP)). After equilibration, the cells were centrifuged and the supernatants were harvested for KinExa kinetic exclusion analysis. In summary, the balanced Jurkat cell supernatants expressing BCMA-55-LS O / SSE CAR containing rhBCMA hFc were eluted onto a streptavidin bead flow cell to capture free biotinylated rhBCMA hFc. The rhBCMA was then detected using a secondary anti-hBCMA antibody that was fluorescently labeled. The absorbance of the detected rhBCMA hFc was recorded for each sample and plotted against the number of cells in each dilution (Darling (2004) Assay Drug. Dev., 2: 647-657). In this study, the Kp for the interaction of cells expressing BCMA-55-LS-O / SSE CAR that bind to rhBCMA hFc in this assay was determined to be approximately 1.46 nM, and the level of expression (EL) was determined to be approximately 146,500 CARs per Jurkat cell that expresses CAR.
[00836] [00836] A membrane proteome disposition (MPA) assay was used to assess the binding specificity of the BCMA-55 derived binding domain, using a scFv-Fc fusion protein. The interactions of BCMA-55-Fc with HEK293 cells that express more than 4400 unique human extracellular proteins, representing more than 85% of the human extracellular proteome and a fluorescent protein were evaluated using the Retrogenix'Y platform. The fluorescent protein was detected to verify transfection, and CTLA4-Fc (tested at 0.2 µg / mL), containing a corresponding Fc, was also used to analyze for CD86 as a positive control. An initial evaluation involved a scFv binding assay for BCMA-55-scFv against the entire panel of proteins. An analysis of the follow-up confirmation was then performed again testing the interaction of
[00837] [00837] Genetically modified human T cells that express several exemplary anti-BCMA CARs were evaluated in vitro after co-culture with target cells expressing BCMA. T cells were transduced with BCMA-52-LS CAR, BCMA-55-LS CAR, BCMA-52-LS-O / SSE CAR or BCMA-55-LS-O / SSE CAR). The responses were compared with cells expressing reference anti-BCMA CAR as a positive control or cells with simulated processing as a negative control. A. Cytolytic activity against target cells
[00838] [00838] Target cells expressing BCMA were incubated with T cells that express the BCMA-52-LS CAR, the BCMA-55-LS CAR or a reference anti-BCMA CAR in an effector-to-target ratio (E: T) of 5: 1, 2.5: 1, 1.25: 1 and 0.65: 1. As a control, the target cells were incubated with T cells not expressing a CAR (simulated control). Specifically, K562 cells transduced by BCMA (K562 / BCMA, BCMAº !! o) or RPMI 8226 cells (human multiple myeloma cell line BCMAºPE *) were used as targets for lysis. Target cells were marked with NucLight Red (NLR) to allow tracking of target cells by microscopy. Cytolytic activity was assessed by measuring the loss of viable target cells over a period of 24 to 72 hours, as determined by a red fluorescent signal (using the Live Cell Analysis System IncuCyte &, Essen Bioscience). The percentage of lysis (% of lysis) was normalized to the lysis that occurred in target cells incubated with simulation-processed T cells. As shown in FIG. 4A, anti-BCMA CAR expression T cells exhibited antigen-specific cytolytic activity against BCMA + cells. The magnitude of cell lysis differed depending on the particular cell line and CAR.
[00839] [00839] In a separate experiment, cytolytic activity was tested with target cells RPMI 8226 at an E: T ratio of 3: 1. As shown in FIG. 4B, cells expressing BCMA-52-LS- and BCMA-55- LS-CAR exhibited approximately 70% of lysis, normalized to lysis by cells processed by simulation not expressing a CAR, while cells expressing the CAR containing the antibody of binding to the reference anti-BCMA antibody showed approximately 50% lysis. Thus, the results showed that the cytolytic activity of cells modified to express BCMA-52- or BCMA-55-CARs was similar to, or greater than, that of the CAR containing the reference binding domain.
[00840] [00840] To compare the cytolytic activity of modified T cells with the same CAR encoded by an unmodified CAR construct or an optimized CAR construct, T cells were modified to express an anti-BCMA CAR using a vector viral containing an unmodified polynucleotide construct (BCMA-52-LS CAR and BCMA-55-LS CAR) or an optimized polynucleotide construct (BCMA-52-LS-O / SSE CAR and BCMA CAR -55- LS-O / SSE). The cytolytic activity of the modified cells was tested substantially as described above. CAR expression T cells were incubated with target cells, K562-BCMA cells, RPMI 8226, MM1.S (human multiple myeloma cell line
[00841] [00841] Cytokine release was assessed after incubation of the various cells expressing anti-BCMA CAR with target cells expressing antigen.
[00842] [00842] Target cells expressing BCMA, K562 / BCMA cells or RPMI 8226, were incubated with T cells expressing the BCMA-52-LS CAR, BCMA-55-LS CAR or an anti-BCMA CAR containing binding domain reference in an E: T ratio of 5: 1, 2.5: 1, 1.25: 1 or 0.6: 1. As a control, the target cells were incubated with T cells not expressing a CAR (simulated control). The cocultivated cells were incubated for about 24 hours and then the supernatants were collected for measurement of IFN-y, TNF-a and IL-2, using a multiplex cytokine immunoassay. As shown in FIG. 5A, the anti-BCMA CAR expression T cells tested produced cytokines after stimulation of the antigen.
[00843] [00843] To assess antigen-dependent cytokine production of modified T cells with the same CAR encoded by an unmodified CAR construct or an optimized CAR construct, T cells were modified to express an anti-BCMA CAR using a vector virus containing an unmodified polynucleotide construct (BCMA-52-LS CAR and BCMA-55-LS CAR) or an optimized polynucleotide construct (BCMA-52-LS-O / SSE CAR and BCMA-55-LS CAR -O / SSE). CAR expression T cells were incubated with target cells, or KS62 / BCMA cells, RPMI
[00844] [00844] Cytolytic activity, cytokine release and proliferation, were evaluated after incubation of BCMA-55-LS-O / SSE CAR expression T cells with BCMA expressing cells that expressed different levels of BCMA. All activity was assessed in the presence or absence of soluble BCMA.
[00845] [00845] “A 1: 1 ratio of primary CD4 + and CD8 + T cells, harvested from two human donors (Dtt1 and Dt2), was stimulated with CD3 / CD28 beads and transduced with a lentiviral vector to stably express the CAR BMCA-55. The transduced cells were cultured in the presence of target cells expressing BCMA at an E: T ratio of 1: 3, 1: 1 or 3: 1. T cells processed by simulation from the same donors were also mixed with the target cells for use as a control. The target cells BCMA +, Daudi, RPMI-8226 and K562-BCMA, exhibited different levels of BCMA antigen density on the surface (antigen density: Daudi (<1000 molecules / BCMA cell) <RPMI-8226 <K562- BCMA) and were stained with succinimidyl carboxyfluorescein ester (CFSE) before incubation with T cells. An equal number of negative target cells
[00846] [00846] BCMA-55-LS-O / SSE TCAR cells exhibited similar cytolytic activity when cultured with target cells, regardless of BCMA expression levels (FIG. 6). In addition, similar results were observed for target cells (NCI-H929) expressing more than 100,000 molecules per cell. The T cells processed by simulation showed no activity against any of the BCMAt target cell lines. Target cells negative for BCMA expression were not lysed by BCMA-55-LS-O / SSE T CAR cells from any of the tested donors (data not shown).
[00847] [00847] Supernatants after incubation were analyzed for the accumulated cytokines of IFN-y, TNF-a and IL-2. The data were consistent with the conclusion that BCMA-55-LS-O / SSE T CAR cells released a range of cytokines after involvement with target cells expressing BCMA; with the level of cytokines released generally corresponding to the increase in the level of antigen (ie, Daudi <RPMI 8226 <K562-BCMA). The results for IFN-y are shown in FIG. 7; similar data were observed for TNF-a and IL-2 (data not shown). CAR BCMA-55-LS-O / SSE T cells did not release cytokines in response to negative BCMA targets, nor did they express cytokines without any target cells present, demonstrating specificity for BCMA + target cells and lack of tonic signaling.
[00848] [00848] BCMA-55-LS-O / SSE CAR expression T cell activity in the presence versus absence of soluble BCMA was evaluated
[00849] [00849] Proliferation in response to BCMA was measured in BCMA-55-LS-O / SSE CAR expression T cells and T cells processed by simulation. Transduced T cells were labeled with cell trace violet (CTV) and cultured in the presence of BCMA positive target cells, BCMA negative target cells or no cells, in a 1: 1 effector to target (E: T) ratio for 72 hours. Proliferation was measured by flow cytometry. T cell proliferation (CD4 + and CD8 + T cells) was observed only for BCMA-55-LS-O / SSE CAR expression T cells in response to incubation with BCMA positive target cells. D. Transduced T cells taken from healthy donors and a patient with myeloma
[00850] [00850] T cells modified to express BCMA-55-LS-O / SSE CARs collected from patients with multiple myeloma were compared to those derived from healthy human donors after a 24-hour incubation with BCMA + and BCMA-K562 target cells. T cells not expressing a CAR were also evaluated as a negative control. CAR T cells derived from patients with multiple myeloma demonstrated similar antigen-specific expression, expansion and expansion activities compared to CAR-expressing cells derived from healthy human donors. Example 8: Anti-BCMA CARs with different spacers.
[00851] [00851] Polynucleotide constructs encoding anti-BCMA CARs were generated that contained different spacer regions between the scFv and transmembrane segments of the encoded CAR polypeptide. Specifically, CARs were generated containing: (1) a spacer derived from an IgG joint region (eg BCMA-5-SS, BCMA-9-SS, BCMA-9-SS, BCMA-18-SS, BCMA-23-SS, BCMA-25 -SS, BCMA-26-SS, BCMA-52-SS, BCMA-55-SS and Reference1 (Vx / VL) -SS); or (2) a short spacer derived from the CD28 ectodomain (for example, BCMA-52-SCD28 and BCMA-55-SCD28). T cells expressing such spacer-containing CARs were compared to T cells transduced with polynucleotide constructs encoding exemplary CARs that contain spacers, as described in Example 3 (eg BCMA-1-LS, BCMA-5-LS , BOCMA-9-LS, BCMA-18 -LS, BCMA-23-LS, BCMA-25-LS, BCMA-26-LS, BCMA-27-LS, BCMA-52-LS, BCMA-55-LS and Reference1 (Vn / VL) -LS).
[00852] [00852] The cells expressing CAR were evaluated for cytolytic activity, monitoring the lysis of target cells of human multiple myeloma of OPM cultured with CAR expression T cells, in an effector to target (E: T) ratio of 1.25: 1 and 0.65: 1. Cells that did not express a CAR (simulation) were used as a negative control. Cytolytic activity was assessed as described in Example 7. For most cells expressing CAR, the lysis of the target cell was greater for cells modified to express a CAR containing a CH2-ChH3 joint spacer compared to the cells modified with a CAR containing a shorter spacer (FIG. 8). Example 9: Agent Assessment of Anti-BCMA CAR Activity Blocking Activity
[00853] [00853] The function of cells expressing anti-BCMA CAR was assessed after incubation with target cells expressing BCMA and soluble BCMA or other proteins. Cytolytic activity, and cytokine production were evaluated substantially as described in Example 7. A. Cytolytic activity
[00854] [00854] T cells expressing anti-BCMA CAR, BCMA-52-LS CAR, BCMA-55-LS CAR or CAR containing the reference binding domain, were incubated with OPM2 target cells in a E: T 5: 1 in the presence of BCMA-Fc soluble at 0, 0.3, 3, 30 or 300 ng / mL. As shown in FIG. 9The cytolytic activity of T cells expressing the CAR containing the BCMA-52-LS reference binding domain or CAR has been substantially reduced in the presence of 3 ng / mL or more of BCMA-Fc, however, the cytolytic activity of cells expressing the BCMA-55-LS CAR was not blocked by the presence of up to 300 ng / mL BCMA-Fc.
[00855] [00855] In another experiment, anti-BCMA CAR expression T cells (BCMA-1-LS CAR, BCMA-9-LS CAR, BCMA-23-LS CAR, BCMA-25-LS CAR, CAR BCMA-26-LS, BCMA-26-LS CAR, BCMA-55-LS CAR and Reference 1 (VW / VL) -LS CAR) were incubated with OPM2 target cells in an E: T ratio 5: 1 in the presence of soluble BCMA-Fc in concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 ng / mL. As shown in FIG. 9B the cytolytic activity of cells expressing BCMA-55-CAR was not blocked by the presence of BCMA-Fc in any of the tested concentrations; however, the presence of varying concentrations of BCMA-Fc blocked the activity of cells expressing other anti-BCMA CARs to different extents.
[00856] [00856] Optimized anti-BCMA CAR expression T cells eliminated from the binding site (O / SSE), BCMA-52-LS-O / SSE CAR, BCMA-55-LS-O / SSE CAR or CAR containing the reference binding domain, were incubated with OPM2 target cells at a 5: 1 E: T ratio in the presence of O, 111, 333 and 1000 ng / mL of culture supernatant from the strain of multiple myeloma cell of H929. The concentration of soluble BCMA was quantified from the H929 supernatant by ELISA. As shown in FIG. 10A the cytolytic activity of cells expressing BCMA-52-LS-O / SSE CAR, BCMA-55-LS-O / SSE CAR or Reference CAR was not blocked by the presence of the H929 supernatant.
[00857] [00857] In an additional study, BCMA-55-LS-O / SSE CAR expression T cells eliminated at the optimized binding site (O / SSE), were incubated with RPMI-8226 tumor target cells in a E: T of 3: 1 in the presence of O, 111, 333 and 1000 ng / ml of soluble BCMA from culture supernatant of the multiple myeloma cell line of H929 (soluble BCMA quantified by ELISA) or BCMA-Fc. The cytolytic activity of cells expressing BCMA-52-LS-O / SSE CAR, BCMA-55-LS-O / SSE CAR or Reference CAR was not blocked by the presence of the H929 supernatant.
[00858] [00858] Optimized anti-BCMA CAR expression T cells pre-eliminated at the binding site (O / SSE), BCMA-52-LS-O / SSE CAR, BCMA-55-LS-O / CAR Reference SSE or CAR, were incubated with OPM2 target cells at an E: T ratio of 5: 1 in the presence of O, 1, 10, 100 and 1000 ng / mL of recombinant B cell activation factor ( BAFF), a binder for BCMA. As shown in FIG. 10B, the cytolytic activity of CAR expression T cells from
[00859] [00859] Anti-BCMA CAR expression cells, BCMA-52-LS CAR, BCMA-55-LS CAR or Reference LS CAR, were incubated with OPM2 target cells in an E: T ratio of 5: 1 in the presence of soluble BCMA. -Fc cells at 0, 111, 333 and 1000 ng / ml. T cells not expressing a CAR (simulated) were also evaluated. The cytokine accumulation of IFN-y, TNF-a and IL-2 in a supernatant was assessed. As shown in FIG. 11A, the accumulation of cytokines in cultures containing T cells expressing the Reference CAR or BOCMA-52-CAR was substantially reduced in the presence of 111 ng / ml or more of BCMA-Fc, however, less reduction in Accumulation of cytokines was observed in cultures containing T cells expressing BCMA-55-CAR in the presence of soluble BCMA-Fc at all tested concentrations.
[00860] [00860] T cells expressing anti-BCMA CAR, BCMA-52-LS CAR, BCMA-55-LS CAR or Reference LS CAR, were incubated with OPM2 target cells in an E: T ratio of 5: 1 in the presence of O, 111, 333 and 1000 ng / ml of culture supernatant from a multiple myeloma cell line of H929. The accumulation of cytokines in cultures containing T cells expressing BCMA-52-CAR, BCMA-55-CAR or Reference CAR was not blocked by the presence of the H929 supernatant (FIG. 11B) Example 10: Antitumor effect of T cells expressing Anti-BCMA CAR after adoptive transfer in vivo in animal model
[00861] [00861] The anti-tumor effects of primary human T cells ex-
[00862] [00862] - NOD.Cg.Prkdc * “lL 2rgt" WiySzJ (NSG) mice were injected intravenously (iv) with 2 x 10º OPM2 cells (multiple myoma) transfected with firefly luciferase (OPM 2-ffluc) ) on day 14, after tumor grafting, the mice received a single intravenous injection (iv) of anti-BCMA T cells expressing (O / SSE) BCMA-23-LS-O / SSE CAR, BCMA CAR -26-LS- O / SSE or BCMA-55-LS-CAR O / SSE eliminated from the binding site, optimized.The anti-BCMA CAR expression T cells were administered in a dose of 1 x 108º (dose low, n = 8) or 3 x 10º (high dose, n = 8) CAR expression T cells per mouse and each condition repeated for CAR expression T cells derived from two different donors. As a control, the mice were cells not expressing a CAR (simulated, n = 8) or were not treated (n = 3), survival and tumor burden were assessed for 90 days.
[00863] [00863] The antitumor activity of the cells expressing the lovingly transferred CAR (CAR-T) was monitored by bioluminescence imaging every 3 to 6 days after the administration of CAR-T cells during the study period. For bioluminescence imaging, the mice received intraperitoneal (i.p.) injections of luciferin substrate (CaliperLife Sciences, Hopkinton, MA) resuspended in PBS (15 µg / g body weight). The mice were anesthetized
[00864] [00864] “As shown in FIG. 12A, for all treated mice, the tumor in mice that received simulated T cells or no T cells continued to develop throughout the study. In comparison to control mice, mice that received an adoptive transfer of modified T cells to express BCMA-23-LS-O / SSE CAR, BCMA-26-LS-O / SSE CAR or CAR BCMA-55-LS-O / SSE were generally observed to have a lesser degree of bioluminescence, indicating a reduction in tumor development over time and / or a lesser degree of tumor development in treated animals. From. The effect on tumor development was greatest with the highest dose of cells expressing anti-BCMA CAR for the exemplary tested anti-BCMA CARs.
[00865] [00865] Survival of mice treated as described above was evaluated and compared until day 79 after the infusion of CAR expression T cells. Representative survival curves, Kaplan-Meier method (GraphPad Prism 7.0, GraphPad Software, La Jolla), from a donor are shown in FIG. 12B. As shown, the anti-BCMA CAR T cells tested at low and high doses resulted in a higher percentage of mouse survival compared to mice that did not receive treatment or T cells processed by simulation. Mice were also evaluated for the presentation of clinical signs associated with tumor burden, including hind limb paralysis (HLP), loss of skin
[00866] [00866] “NOD.Cg.PrkdescidIL2rgtm 1WilVSzJ (NSG) mice were injected subcutaneously with RPMI 8226 cells (peripheral blood plasmacytoma). On day 27, the mice were classified into groups based on a minimum mean tumor volume of approximately 130 mm . On day 29, the mice received a single intravenous (iv) injection of primary human T cells (CD4 + and CD8 +) modified to express BCMA-23-LS-O / SSE CAR, BCMA-26-LS CAR -O / SSE or CAR of BCMA-55-LS- O / SSE eliminated at the union site, optimized (O / SSE) in a dose of 1 x 10º (low dose, n = 8) or 3 x 10º (high dose , n = 8) CAR expression T cells. Each condition was repeated for CAR expression T cells derived from two different donors. Mice that were given cells that were processed by simulation and untreated mice were used as negative controls. The volume of the tumor was measured by calipers twice a week until day 152 after the transfer of T CAR cells and the mice were euthanized when dying, 20% weight loss or when the volume of the tumor exceeded 1500 mm . Survival curves were plotted until day 108 after T CAR cell transfer using the Kaplan-Meier method (GraphPad Prism 7.0, GraphPad)
[00867] [00867] “Representative results for tumor development and survival of donor-derived CAR expression T cells are shown in FIGs. 13A and 13B, respectively. As shown in FIG. 13A, the tumor continued to develop throughout the study after the adoptive transfer of negative control cells or in mice that did not receive treatment. In comparison to control mice, mice that received an adoptive transfer of T cells modified to express BCMA-23-LS-O / SSE CAR, BCMA-26-LS-O / SSE CAR or BCMA-55 CAR -LS-O / SSE showed a substantially reduced tumor volume after receiving the low or high dose of T cells expressing CAR (FIG. 13A). In this model, the mice that were administered both tested doses of anti-BCMA T CAR cells exhibited complete regression of tumor development 20 days after the transfer of T CAR cells, which continued throughout the duration of the study evaluation shown in FIG . 13A.
[00868] [00868] The survival percentage of the mice that were administered anti-BCMA CAR expression T cells was also substantially higher than the control groups (FIG. 13B). At 108 days after the infusion of CAR T cells, two animals had lost post-tumor elimination in the group treated with the high dose of BCMA-26-LS-O / SSE CAR expression T cells, although this was probably caused by the symptoms of graft versus host disease (GVHD) in this model. All other mice treated with T CAR cells remained alive until 108 days after the administration of T T cells.
[00869] [00869] The presence of CAR + T cells in the blood was monitored to assess the pharmacokinetics of CAR expression T cells in the treated mice. The 8 mice in each treatment group were divided into 2 groups of 4 mice. Blood was collected weekly, by retro-orbital bleeding, alternating between the 2 groups, so that each mouse was bled every two weeks for 4 weeks after the administration of T CAR cells (that is, on days 7 , 14, 21 and 28 after administration of
[00870] [00870] The number of CD4 + and CD8 + T cells per ul of blood on days 7, 14, 21 and 28 is shown in FIG. 14A and FIG. 14B, respectively, for a donor and in FIG. 15A and FIG. 15B, respectively, for the second donor. As shown, CAR-T expansion occurred in groups of high and low doses in CD4 + and CD8 + T cells, with maximum or peak expansion observed on day 14 after the transfer of CAR T cells to both donors. At all times evaluated after CAR-T cell transfer, a greater number of CAR + CD8 + T cells were observed compared to CAR + CD4 + T cells for both donors (compare FIG. 14A and FIG. 14B or FIG. 15A and FIG. 15B). T cells modified to express BCMA-55-LS-O / SSE CAR exhibited higher CAR expression compared to BCMA-23-LS-O / SSE CAR expression T cells and BCMA-26-LS CAR constructs -O / SSE, which showed expression comparable to each other. These results demonstrate that BCMA-55-LS CAR expression T cells can be identified circulating in the blood during tumor clearance. Example 11: Evaluation of signals by the chimeric anti-BCMA antigen receptor (CAR) on a reporter signal from Nur77-tdTomato on Reporter Cell Line
[00871] [00871] An exemplary stable Jurkat T cell reporter cell line containing a Nur77 insertion reporter was generated, where the nucleic acid sequences encoding the reporter molecule were inserted into the endogenous Nur77 locus by means of dependent repair. of homology (HDR). The orphan nuclear hormone receptor
[00872] [00872] Viral vectors containing polynucleotides that encode the following anti-BCMA chimeric antigen receptors (CARs), described in Example 3, were introduced into the Jururat T cell line of reporter Nur77-tdTomato: BCMA-55-LS- CAR O / SSE, BCMA-26-LS-O / SSE CAR, BCMA-23-LS-O / SSE CAR and BCMA-25-LS-O / SSE CAR. Reporter cells expressing anti-BCMA CAR were evaluated for Nur77 signaling activity in response to increasing amounts of plaque-bound recombinant BCMA or in response to exemplary multiple myeloma cell lines after 20 hours of co-culture. A. Nur77 signaling in response to recombinant BCMA | attached to the plate
[00873] [00873] Reporter cells transduced with a viral vector encoding CAR of BCMA-55-LS-O / SSE were incubated for 6 hours in 96-well cell culture plates that were coated overnight with varying concentrations ( 0.008 µg / ml, 0.04 µg / ml, 0.2 µg / ml, 1 µg / ml and 5 µg / ml) of BCMA-Fc fusion polypeptide (soluble human BCMA fused at its C-terminal to an Fc region IgG). A recombinant Fc polypeptide was used as a control (Control Fc). As shown in FIG. 16A, a dose-dependent increase in tdTomato expression was observed after stimulation of reporter cells expressing anti- <BCMA CAR with recombinant antigen.
[00874] [00874] In another study, reporter cells modified to express BCMA-55-LS-O / SSE CAR, BCMA-26-LS-O / SSE CAR, BCMA-23-LS-O / SSE CAR and BCMA-25-LS-O / SSE CAR were incubated with ten (10) 2-fold serial dilutions of BCMA-Fc. Reporter cells expressing an anti-CD19 CAR were used as a non-target control. The percentage of cells expressing tdTomato in the population of cells expressing CAR was determined (as determined based on the expression of the substitute marker). As shown in FIG. 16B, a dose-dependent increase in tdTomato expression was observed after stimulation of the recombinant antigen. No response to BCMA-Fc stimulation was observed by control reporter cells that express a CAR against a non-target antigen. B. Nur77 signaling in response to multiple myeloma cell lines
[00875] [00875] Reporter cells transduced with a viral vector that co-replicates BCMA-55-LS-O / SSE CAR were incubated for 20 hours with NALM6, Daudi, RPMI-8226, MM1S, OPM2 and H929 cells. Different levels of RFP expression were observed depending on the cell line that provided stimulation of the anti-BCMA CAR expression reporter cells.
[00876] [00876] To assess the amounts of BCMA expression on the surface of multiple myeloma cell lines used to stimulate the anti-BCMA CAR expression reporter cells, the cells were stained with anti- <BCMA antibody (BioLegend, San Diego, CA), flow cytometry events were collected on an LSRFortessa "y flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed using FlowJo software (Treestar Inc., Ashland, OR) The density of BCMA (AD) antigen was determined using quantum anti-mouse IgG microsphere beads beads “Simply Cellular & coated with the same anti-human BCMA antibody. The microspheres were labeled, and the ability to | li - BCMA antibody generation was calculated The results confirmed the detection of a parameter (detectable reporter levels) indicative of specific CAR activity in CAR expressing reporter cells, when incubated with each of several different expression cells of BCMA, exhibiting a range of different antigen densities, and not when incubated with target-negative cells. The degree of the RFP reporter signal generally correlates with BCMA surface expression levels. When incubated with cells in which lower levels of surface BCMA expression were observed, the CAR expression reporter cells exhibited lower levels of the reporter indicative of activity. Likewise, CAR expression reporter cells incubated with cell lines in which higher levels of
[00877] [00877] The reporter's expression in cells modified to express anti-BCMA CARs containing the same antigen-binding domain, but spacers of different lengths was determined after co-culture with target cells. Jurkat Nur77-tdTomato cells, generated as described in Example 11, were modified to express BCMA-55-LS-O / SSE CAR (containing a longer spacer derived from modified IgG-Cn2-CH3, mentioned in SEQ ID NO: 649) or BCMA-55-SS CAR (containing a shorter spacer derived from the I9G4 joint, mentioned in SEQ ID NO: 363). The cells were co-cultured with K562 target cells expressing human BCMA (BCMA-K562) in various E: T ratios. Reporter cells expressing a CAR directed to a different antigen (CAR anti-CD19) were used as a control. As shown in FIG. 17, it was observed that the expression level of Nur77-tdTomato was different in anti-BCMA CARs containing different lengths of spacer, and a dose-dependent response was observed for stimulation with target cells that expressed
[00878] [00878] Nur77-tdTomato reporter cells were transduced with a viral vector encoding anti-CD19 CAR (control), BCMA-55-LS-O / SSE CAR, BCMA-26-LS-O / SSE CAR, CAR BCMA-23-LS-O / SSE, or BCMA-25-LS-O / SSE CAR, as described in Example 11 above, with the exception that the substitute marker for transduction was a super-duplicated green fluorescent protein, sSÍGFP . In this model, tonic signaling was indicated by the expression of tdTomato in the absence of BCMA antigen stimulation.
[00879] [00879] A viral vector encoding an anti-BCMA CAR containing a different anti-BCMA scFv, designated as BCMA-52-LS-O / SSE CAR, was also generated and transduced in the reporter cell. The various cells expressing CAR were incubated without antigen stimulation to assess the degree of antigen-independent signaling for 3 days and evaluated for tdTomato expression by flow cytometry.
[00880] [00880] “As shown in FIG. 18, several CAR expression cell lines exhibited a variable degree of tdTomato expression in the absence of antigen stimulation. The percentage of tdTomato + cells (indicative of tonic reporter activation) among cells expressing CAR (indicated by GFP + cells) ranged from 0.23% to 19.3%, in cells expressing different CARs. Example 14: Evaluation of antigen-independent signaling (tonic) from chimeric anti-wBCMA antigen receptors (CARs) containing different intracellular domains
[00881] [00881] Antigen-independent signaling (tonic) has been evaluated in reporter cells that express several CARs containing different
[00882] [00882] “As shown in FIG. 19A and FIG. 19B, intracellular domains derived from 4-1BB and CD28 in various CARs resulted in different levels of tonic signaling, as indicated by the percentage of tdTomato + cells among CAR + cells (as determined based on the expression of the substitute marker ). Example 15: Evaluation of antigen cross-reactivity of chimeric anti-BCMA antigen receptors (CARs) using the reporter cell line
[00883] [00883] The Nur77-tdTomato cell line modified to express BCMA-55-LS-O / SSE CAR, specific for human BCMA and generated as generally described in Example 11, was employed to assess cross-reactivity of species of the CAR antigen binding domains. The BCMA-55-LS-O / SSE CAR expressing reporter cell line was cultured with K562 human myeloid leukemia cells expressing human BCMA (huBCMA), murine BCMA (muBCMA) or cynomolgus monkey BCMA (cynoB-CMA) , in an E: T ratio of 2: 1 or 5: 1. The percentage of tdTomato + cells was determined by flow cytometry.
[00884] [00884] “As shown in FIG. 20A, more than 90% of the cells
[00885] [00885] —Reporter cell alignment expressing BCMA-55-LS-O / SSE CAR was incubated with increasing concentrations (0, 0.1, 0.25, 1, 2.5, 10, 25 and 100 ug / mL) of huBCMA and cynoBCMA coated in 96-well flat-bottom plates. The percentage of tdTomato + cells, and the mean fluorescence intensity (MFI) of the tdTomato signal in the CAR + cells were determined.
[00886] [00886] As shown in FIGS. 20B and 20C, cynoBCMA did not cross-react with BCMA-55-LS-O / SSE CAR at low concentrations, but at high concentrations. Example 16: Evaluation of antigen specificity of anti-BCMA chimeric antigen receptors (CARs) using reporter cell lineage
[00887] [00887] The antigen specificity for cell activation expressing BCMA-55-LS-O / SSE CAR was tested by comparing the activation of Jurkat Nur77 reporter cells in response to BCMA-expressing MM1S target cells, with target cells of K562 modified to express a non-BCMA protein demonstrated to be recognized at low levels by BCMA-55-scFv Fc in Example 5C, Cathepsin G (CTSG). As a negative control, parental K542 cells were also evaluated. In summary, Nur77 reporter cells, transduced with a viral vector encoding BCMA-55-LS-O / SSE CAR, were incubated 24 hours with the target cells listed above, in relation to
[00888] [00888] - Epitopes recognized, for example, specifically linked to, by exemplary anti-BCMA scFv clones (BCMA-1, BCMA-5, BCMA-9, BCMA-23, BCMA-25, BCMA-26 scFvs, BCMA-52 and anti-BCMA from BCMA-55), were evaluated using complete discontinuous epitope mapping by Chemical Linkage of Peptides on Scaffolds (CLIPS; Pepscan Presto BV, Lelystad, Netherlands; see, for example, Timmerman et al ., (2007) J. Mol. Recognit. 20: 283-329). Mapping was performed using anti-BCMA scFv clones, such as those fused with mouse Fc (scFv-mFc).
[00889] [00889] Linear and conformational peptide libraries of human BCMA amino acid residues 1-54 (mentioned as amino acid residues 1-54 of SEQ ID NO: 367) were generated based on a combinatorial matrix design. Linear and structural mimetic peptides, including single loop imitations, a-helix, B-cycle, combinatorial and linear disulfide bridge mimics, discontinuous epitope mimics, along with positive and negative control peptides, on a functionalized solid support per amino.
[00890] [00890] Affinities for binding to peptides in the epitope library were determined using ELISA. The peptide matrices were incubated with a solution containing scFv overnight at 4ºC. Affinity information was used in iterative analyzes to determine
[00891] [00891] The scFvs evaluated were observed for recognized conformational epitopes that included several discontinuous peptide stretches of the BCMA peptide sequence. BCMA-1, BCMA-5, BCMA-23 and BCMA-25 scFv were observed to bind to a 36SNTPPLTCQR; 39 peptide (mentioned in SEQ ID NO: 379), which can be recognized in a linear fashion. In some respects, such antibodies recognize a non-linear or linear epitope, including residues of such a peptide of SEQ ID NO: 379, and in some respects, to recognize a non-linear epitope, also including residues of 2; CIPCQLR> 27 (mentioned in SEQ ID NO: 375) 3SNTPPLTCQR3s and / or 448 VTNSVK5o (mentioned in SEQ ID NO: 393). BCMA-26 scFv was found to recognize an epitope comprising residues present in gCSQNEYF14 (mentioned in SEQ ID NO: 410) and 17LLHACIPCQLR> 27 (mentioned in SEQ ID NO: 428). BCMA-52-scFv-mFc was observed to bind to an epitope containing residues of the following discontinuous peptides: 10QNEYF14 (SEQ ID NO: 637), 21CIPCQL26 (SEQ ID NO: 638) and; rCQARYC4 (SEQ ID NO: 639) . It was observed that BCMA-55-scFv-mFc specifically binds to an epitope containing residues present in peptides that comprise discontinuous portions of the BCMA polypeptide sequence, comprising the following sequences individually:; »« MLMAGs (SEQ ID NO: 640), 13Y FDSL17 (SEQ ID NO: 779) and 25sQLRCSSNTPPL35 (SEQ ID NO: 642). In some embodiments, the antibody or receptor provided specifically binds to an epitope that comprises residues present in one or more of, for example, each of the batch peptides having the sequences of: MLMAG (SEQ ID NO: 640) , YFDSL (SEQ ID NO: 779) and QLRCSSNTPPL (SEQ ID NO: 642). In some respects, the antibody or receptor provided specifically binds to an epitope comprising residues present in one or more of, for example, each of the following discontinuous peptides having the sequences of: MLMAG (SEQ ID NO: 640), YFDSLL ( SEQ ID NO: 641) and QLRCSSNTPPL (SEQ ID NO: 642); in some respects, the provided antibody or receptor specifically binds to an epitope comprising residues present in one or more of, for example, each of the following discontinuous peptides having the sequences of: MLMAG (SEQ ID NO: 640 ), QONEYFDSLL (SEQ ID NO: 780) and QLRCSSNTPPL (SEQ ID NO: 642). Example 18: Administration of cells expressing anti-BCMA CAR to individuals with relapsed or refractory multiple myeloma (MM)
[00892] [00892] T cell compositions expressing a chimeric antigen (CAR) receptor containing autologous T cells that express a specific CAR for B cell maturation antigen (BCMA) were administered to human subjects with multiple myeloma (MM ) relapsed and / or refractory. A. Individuals and Treatment
[00893] [00893] Compositions containing autologous T cells modified to express an exemplary BCMA-specific CAR were administered to adult human subjects with relapsed or refractory multiple (MM) myeloma, who received 3 or more previous treatments (the 3 or more previous treatments, including at least one proteasome inhibitor, an immunomodulatory agent and an anti-CD38 monoclonal antibody, in each case, unless the individual was not a candidate for such treatment, as contraindicated) .
[00894] [00894] The T-cell compositions administered were generated by a process that includes immuno-affinity enrichment of the CD4 + and CD8 + cell populations of leukocyte samples.
[00895] [00895] Two to seven days before the infusion of CAR + T cells (and completed at least 48 hours before the infusion of CAR-T) the subjects received a lymphodepletary chemotherapy (LDC) with fluridabine (flu, 30 mg / m / day) and cyclophosphamide (Cy, 300 mg / m2 / day) for 3 days, LDC completed at least 48 hours before CAR-T infusion. The cryopreserved cell compositions were thawed at the bedside prior to intravenous administration, with the day of infusion being designated day 1. On day 1, subjects received a dose of CAR expression T cells, as follows: one single dose level 1 (DL1) containing 5 x 10 7 T cells of total CAR expression, or a single dose 2 dose (DL2) containing 1.5 x 10 T cells of total CAR expression.
[00896] [00896] At one point in the analysis, 19 adult individuals were included in an ongoing clinical study involving such therapy. Of these 19 individuals at that particular time, 13 individuals were administered with anti-BCMA CAR + cells, each in DL1 or DL2. Of these 13 individuals, at this particular moment in the ongoing study, 8 individuals were assessed for indicative safety attributes (assessment based on> 1 month of follow-up) (n = 5 DL1; n = 3 DL2). One individual was unable to receive CAR + T cells due to sepsis after LDC, leading to death before CAR + T cell administration. Three individuals (all DL1) were evaluated at this time for the confirmed response (evaluation based on> 2 months of follow-up) according to the uniform response criteria of the International Myeloma Working Group (IMWG) (Kumar et a /. ( 2016) Lancet Oncol 17 (8).): E328-346).
[00897] [00897] For these 8 individuals evaluated at this time, the average follow-up was 5 weeks (range, 4 to 13 weeks). The mean age was 53 years (range 36 to 66), with an average time since diagnosis of 4 years (range 2 to 12). Individuals received an average of 10 previous regimens (range 4 to 15) for MM. Of the 8 individuals, 4 (50%) were refractory (no response or progression within 60 days of the last therapy) to bortezomib, carfilzomib, lenalidomide, pomalidomide and an anti-CD38 monoclonal antibody. Seven of the 8 individuals (88%) had already undergone autologous stem cell transplantation and 4 of 8 (50%) had high-risk cytogenetics for IMWG.
[00898] [00898] “At the time of the evaluation at the time of the study being carried out, no DLT was observed in the evaluated individuals who received DL1 or DL2. Cytokine release syndrome (CRS), all grades 1 or 2, was observed in 6 of the 8 (75%) individuals at the time. The median onset of CRS at the time among the 8 individuals was 9 days (range 4 to 10), with an average duration of 4.5 days (range 2 to 19 days). None of the individuals with grade 2 CRS at the time required vasopressor support and only 1 individual received tocilizumab. None of the individuals had CRS grade 3 or greater. Three of the 8 (38%) individuals had neurological adverse events (LA). Two of the eight individuals at the time had grade 1 events and 1 had a grade 3 event (lethargy), which resolved within 24 hours after receiving steroids. The onset of neurological AEs was 9, 11 and 12 days, lasting 2, 3 and 1 days, respectively, for the 3 individuals who presented neurological AE. The individual who experienced neurotoxicity (NT) grade 3 at the time of the analysis had developed secondary plasma cell leukemia (PCL) just before receiving LDC.
[00899] [00899] “It was observed that all 8 individuals at the time presented evidence of an objective response, including the individual with secondary PCL. It was observed that three individuals, all administered DL1, obtained confirmed responses (1 partial response, RP; 2 strict complete responses, SCR), while the others remained unconfirmed (1 complete response, CR; 2 very good partial responses, VGPR; 1 PR, 1 MR). Up to the time of the evaluation, no individual had progressed.
[00900] [00900] The results showed that, at the dose levels assessed, the administration of anti-BCMA CAR cell therapy exhibited favorable safety profiles, with no DLTs reported at this point in an ongoing clinical study. The results were consistent with the conclusion that, at this time, the incidence of NT grade 3 or greater was low, and no CRS grade 3 or greater was observed with clinical response.
[00901] [00901] The present invention should not be limited in scope to the particular described modalities, which are provided, for example, to illustrate various aspects of the invention. Several changes in the described compositions and methods will become evident from the description and the teachings contained herein. Such variations can be practiced without departing from the true scope and spirit of the invention and must be included in the scope of the present invention.
BCMA-1, -3, - 4, -5, -6, -7, -8, -25, -31, -1, DYAMS 42, -44, -45, -46, -51 (CDR-H1 SEQUENCES) aa) BCMA-2, -9, - 10, -12, -17, -21, -22, -23, -24, -26, -32, -33, -35, - Kabat CDR-H1 numbers 36, - pYYMS 37, -38, -40, -41, -47, -48, - 49 (aa) BCMA-11, -20, 3, Kabat CDR-H1 numbering DYAMH -27, -28, -29, -30 , -34, -39 (aa) BCMA-1, -3, - 4, -5, -6, -7, -8, -25, -31, -4, Kabat CDR-H2 numbering, - 4 FIRSKAYGGTTEYAASVKG 42, - 44, 45, -46, -61 (aa) BCMA-2, -9, - 10, -12, -17, -21, -22, -23, Kabat CDR-H2 numbering - YISSSGSTIYYADSVKG 26, -32 , -35, -36, -37, -38, - 40, -47, -48, -49 (aa) BCMA-11 Kabat CDR-H2 numbering, -20, GISWNSGSIGYADSVKG -24, -27, -29 , -30, -34, -39 (aa) BCMA-1, -3, -3, -3, -4, -5, -6, -7, -8, -25, -31, -7, Kabat CDR-H3 numbering 7 WSAPTDY 42, -44, -45, -46, -51 and Vir 2 (aa) | 8 | vpePPSSDI BCMA-2 CDR-H3 (aa) BCMA-9, -36, CDR-H3 CDR-H3 37, -38, -41 and Vu-8 (aa) BCMA-10, -12, VDGPPSFDI CDR-H3 -26, -32, -47, -48, 49 and V- 1 (aa) 1 BCMA-11 DLGPDYDPDAFDI CDR-H3, -34 (aa) BCMA-1 CDR-H1, -3, - GFTFGDY 4, -5, -6, -7, -8, -25, -30, -
ee - Toe 31, -42, -44, -45, -46, -51 (aa) BCMA-2, -9, - 9, - Chothia CDR-H1 numbering, -9, - 12, -17, -21, -22, -23 , -24, - 26, -32, -33, -35, -36, -37, - B GFTFSDY 38, -40, -41, -47, -48, -49 (aa) Chothia CDR-H1 numbering BCMA-10 (aa) BCMA-11, -20, GFTFDDY -27, -28, -29, -34, -39 (aa) BCMA-1, -3 Chothia CDR-H2 numbering, - 4, -5, -6, -7, -8, -25, -31, - 16 RSKAYGGT 42, -44, -45, -46, -51 (aa) BCMA-2 Chothia CDR-H2 numbering , -9, - 10, -12, -17, -21, -22, -23, - 17 SSSGST 26, -32, -35, -36, -37, -38, - 40, -47, -48 (aa) BCMA-11, -20, 18 SWNSGS Chothia CDR-H2 numbering -24, -27, -29, ão, -34, -39 (aa) BCMA-1, Chothia CDR-H1 numbering, - 3, - 4, -5, -6, -7, -8, -25, -31, - 19 GFTFGDYAMS 42, -44, -45, -46, -51 (aa) ADM numbering BCMA-2, - 9, -12, -17, -21, - | 22, -23, -24, -26, -32, -33, - GFTFSDYYMS 35, -36, -37, -38, -40, -41, - 47, -48, -A9 CDR-H1 from (aa ) BCMA-10 APM CDR-H1 numbering (aa) BCMA-11 CDR-H1, -20, 22 GFTFDDYAMH -27, -28, -29, -34, -39 (aa) ADM numbering
BCMA-1, -3, -4, -5, -6, -7, -8, -25, -31,-CDR-H2, 23 FIRSKAYGGTTE 42, -44, -45, -46, 51 (yy) BCMA-2, -9, - 10, -12, -17, -21, -22, -23, -24, ADM CDR-H2 numbering YISSSGSTIY 26, -32, -35, -36, -37, - 38, - 40, -47, -48, -49 (aa) BCMA-11, -20, GISWNSGSIG -24, -27, -29, -30, -34, -39 (aa) CDR-H2 ADM numbering ) BCMA-1 APM number KSSQSVLSTSNNKNYLA CDR-L1 (aa) BCMA-2 RASQSIKTNLA CDR-L1 (aa) BCMA-S, -46 RASQDIRNSLA CDR-L1 from BCMA-4 (KSSQSVLHSSNNKNYL) BCMA-S, -8, - 24 (aa) BCMA-6 RASQSISNSLA CDR-L1 (aa) BCMA-7 RASQDIGDYLA CDR-L1 (aa) 33 BCMA-S GANNIGSKSVH CDR-L1, -26, - ( aa) BCMA-11 SGSSSNIGSNAVN CDR-L1 (aa) BCMA-12 SGSRSNIGNNYVS CDR-L1 (aa) BCMA-3, 5, CDR-L2, WASTRES 90144 46 (0o) to BCMA-4 AASRLES CDR-L2 , 42 (aa) BCMA-7 VASTLOS CDR-L2 (aa) a3 BCMA-9 DDDDRPS CDR-L2, -26, - 35 (aa)
BCMA-1 QQYFSSPYT CDR-L3 (aa) BCMA-3 CDR-L3 (aa) QQYYSSPLT CDMA-3 CDR-L3, -46 (aa) BCMA-4 QRYYSLPLS CDR-L3 (aa) QQSHMYPPT CDR- BCMA-6 L3 (aa) BCMA-7 QQYHSHPWT CDR-L3 (aa) BCMA-8 QQYYSTPYT CDR-L3, -31 (aa) BCMA-10 QAWDSSSTLYV CDMA-10 (aa) AAWDDSLRGYV CDR-L3 from BCMA -11 (aa) 58 BCMA-12 QUWDSSSDHWV CDR-L3, -32, -48 (aa)
59 QVALVASGEGGLVQPGRSLRLSCTASGFT | BC1 Vx of BCMA-1, -3, 4, FG -5, -6, -7, -8, -45, -46 (aa) EVOLVESGGGLVKPGGSLRLSCAASGFT | BC1-FRY V1, -12, - FS 22, -23, -26, -40, -48 (aa) EVOLVAOSGGGLVKPGGSLRLSCAASGFT | BCMA-S FR1 Vy, -24, -
61 FS 35, -37 (aa)
EVALVESGGELVKPEGSLRLSCAASGFP FR1 Vu of BCMA-10 (aa)
63 QVALVASGGGLVQPGRSLRLSCAASGF | BCMA-11 FR1 Vu, -28, TFD -29, -39 (aa)
BCMA-1 FR2 Vu, -3, 44,
64 WFROAPGKGLEWVG -5, -6, -7, -8, -25, -31, -42,
44, -45, -46, -51 (aa) FR2 Vx BCMA-2, -9, -10, - 12, -21, -22, -23, -24, -26, - 65 WIRQAPGKGLEWVS 32 33/35 , -36, .37 38. 40, -41, -47, -48, -49 (yy) RETISRDDSKSIAYLQMNSLKTEDTAVYY | FR3 BOMA-1, -3, 4, 67 CAR -5, -6, -7, -8, -25, -31, -42, 44, -45, -46, -51 (aa) RFTISRDNAKNSLYLQaMNSLRAaEDTAVYYy | R $ Vu de BOMA-2, -10, - CAK 12, -21, -22, -23, -26, -32, - 40, -41, -48, -49 (aa)
RFTISRDNAKNSLYLQMNSLRAEDTAVYY | BC $ FR of BCMAZS 11 - CAR 17, -24, -28, -29, -30, -33, - 34, -35, -36, -38, -39 (aa) BCMA-1 FR4 Vu, - 3, 4, -5, -6, -7, -8, -9, -15, -16, - 18, -20, -21, -22, -23, -25, - 7O WGQGTLVTVSS 27 31 35, 36, .37 38. 40, -41, -42, -44, -45, -46, - 51 (aa) BCMA-2, -10, -10, -11, -12, -24, -26, BC4-Vu -29, -30, - Ti WGQGTMVTVSS 32. 34 39, 48.49, 50 (aa) AIRMTQSPSSLSASLGDRVTITC FR1 V. of BCMA-4 (aa) DIVMTOSPDSLAVSLGERATINC the VI of BCMA-S, -8, -24 VIQLQLOSDRSVSMAV -7 (aa) SYELTQPPSVSVAPGQTARVTC FR1 V. of BCMA-9 (aa) [1% SYVLTQPPSVSVAPGQTARITC .s Vi of BOMA-10, 26 QLVLTQPPSASGTPGORVTISC FR1 V. of BCMA-11 (aa)
ASALTOPPSVSARPGAKVTISC WYQQKPGQPPRLLLY FR2 V. of BCMA-1 (aa) WYQQKPGQAPRLLIY the Vi of BEMA-2, 34 FR2 V, of BCMA-3, -5, 8, WYQQKPGQPPKLLIY 24,44, 46 (aa) 86 | wYooRPGKAPKLLLS FR2 V. of BCMA-4 (aa) WYKQRPGEAPKLLIH FR2 V. of BCMA-6 (aa) WFQQRPGKAPKSLIY FR2 of V. BCMA-7 (aa) WYQQKPGQAPMLVVY FR2 Vi of BCMA-9, -26, - (aa) wraaKPeaaavPWY WYQQLPGTAPEVLIY FR2 V. of BCMA-11 (aa)
WYQQLPGTAPKLLIY FR2 V. of BCMA-12 (aa) 93 GVPDRFSGSGSGTDFTLTISSLQAEDVAV | FR3 V, BCMA-1, -3, 5, YYc -8, -31, -44, -46 (yy) 94 GIPDRFSGSGSGTDFTLTITRLEPEDFAV | n53 y, by BCMA-2 (az) YYc GVESRFSGTTSGAEYALSISSLQPEDVAS FR3 Vi by BCMA-A (as) GVPSRFSGRGSGTVFTLAISNVOPEDFA | 253, BCMA-6 (aa)
TYYC 97 GVPSRFSGSGSGTHFTLTINSLOPEDFAT | ce3 v, BCMA-7 (aa) YYc GIPERFSGSNSGNTATLTISGVEAGDEAD | FR3 V. of BCMA-9, -26, - YFC 35 (aa) GIPERFSASNSGNTATLTISGAGATDEAE | n53 y, BCMA-10 (aa) YYc 100 GVPDRFSGSKSGTSASLAINGLOSEDEA | 573 y, qe BCMA-11 (aa) DYYc 101 GIPDRFSGSKSGTSATLDIAGLOTGDEAD | re3 v, of BCMA-12 (aa) YYc FGHGTKLEIK FR4 V. of BCMA-1 (aa) 103 FGRGTKLEIK FR4 V. of BCMA-2, -39 (aa) FR4 V. of BCMA-3, -4, 6, FGGGTKVEIK 20, -46 (sm) 105 FGQGTKVDIK FR4 V. of BCMA-5, -45 (aa) 107 FEQGTKLEIK FR4 V. of BCMA-8, -44 (aa) FR4 V, of BCMA-9, -10, - FR4 V. of BCMA-12, -14, - 15, -16, -17, -18, -32, -33, - 109 FGGGTKLTVL 36-37 -38, -41 48, .49.- 50 (aa) QVALVASGGGLVAPGRSLRLSCTASGFT: 110 FGDYAMSWFRQAPGKGLEWVGFIRSKA ao YGGTTEYAASVKGRFTISRDDSKSIAYLQ | TOS O MNSLKTEDTAVYYCAAWSAPTDYWGQG
Eee es the EV EVOQLVESGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYISSSGS 111 TIYYADSVKGRFTISRDNAKNSLYLQMNS | BCMA-2 Vu Chain (aa) LRAEDTAVYYCAKVDGPPSSDIWGQGTM VTVSS EVOLVOSGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYISSSGS 112 | TIYYADSVKGRFTISRDNAKNSLYLQMNS | BCMA-S9 Vu Chain (aa) LRAEDTAVYYCARVDGDYVDDYWGQOGT LVTVSS
EVOLVESGGGLVKPGGSLRLSCAASGFP FSDYYMSWIRQAPGKGLEWVSYISSSGS | 113 TIYYADSVKGRFTISRDNAKNSLYLQMNS | BOMA-10 Chain Vu LRAEDTAVYYCAKVDGPPSFDIwGEGTM | (92) VTVSS
QVALVOSGGGLVOPGRSLRLSCAASGF TFDDYAMHWVRRAPGKGLEWVSGISWN | 114 SGSIGYADSVKGRFTISRDNAKNsLYLaM | Va chain of BOMA-11 NSLRAEDTAVYYCARDLGPDYDPDAFDI | (92) WGQGTMVTVSS
EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS: 115 TIYYADSVKGRFTISRDNAKNSLYLQMNS from ca) Va BOMA-12, -286, - LRAEDTAVYYCAKVDGPPSFDIWGQGTM VTVSS
DIVMTQSPDSLSVSPGERATISCKSSQSV LSTSNNKNYLAWYQQKPGQPPRLLLYW: 116 ASTREAGVPDRFSGSGSGTDFTLTISsLO | Chain V. of BOMA-1 (aa)
AEDVAVYYCQQYFSSPYTFGHGTKLEIK EIVMTQOSPATLSVSPGETATLSCRASOS! KTNLAWYQQKPGQAPRLLIVAASTRATGI | nm PDRFSGSGSGTDFTLTITRLEPEDFAVYy | Chain Vi of BOMA-2 (aa) CQAQYGSSPTFGRGTKLEIK
DIVMTQSPDSLVVSLGERATINCKSSQSV 118 - | LHSSNNKNYLAWYQQKPGQPPKLLIVWA) Chain V, BCMA-3, -46 STRESGVPDRFSGSGSGTDFTLTISSLOA | (aa) EDVAVYYCQQYYTTPLTFGGGTKVEIK
AIRMTOSPSSLSASLGDRVTITCRASQDI 119 - | RNSLAWYQQRPGKAPKLLLSAASRLESG | BCMA-4 Chain Vi (aa) VPSRFSGTTSGAEYALSISSLQPEDVASY
Fa es | | FEAOYYSLPLSFGGETKVEIK |
DIVMTQSPDSLAVSLGERATINCKSSQSV LYSSNNKNYLAWYQQKPGQPPKLLIYWA: 120 STRESGVPDRFSGSGSGTDFTLTISSLOA | Chain V. of BOMA-S (aa)
EDVAVYYCQQYYSTPWTFGQGTKVDIK DIVMTQSPSSLSVSVGERVTITCRASOQS! SNSLAWYKQRPGEAPKLLIHAASNVEDG: 121 VPSRFSGRGSGTVFTLAISNvOPEDFATY | BCMA-6 Chain V. (aa)
YCQASHMYPPTFGGGTKVEIK VIQLTASPSSLSASVGDRVTITCRASQDI! GDYLAWFQQRPGKAPKSLIYVASTLOSG: 122 VPSRFSGSGSGTHFTLTINSLQPEDFATY | Chain V. of BOMA-7 (aa) YCQQAYHSHPWTFGPGTKVDIK
DIVMTQSPDSLAVSLGERATINCKSSQSV LYSSNNKNYLAWYQQKPGQPPKLLIVWA: 123 STRESGVPDRFSGSGSGTDFTLTISSLOA | BOMA-8 V chain (aa) EDVAVYYCQQYYSTPYTFGOGTKLEIK
SYELTOPPSVSVAPGOTARVTCGANNIG SKSVHWYQQKPGQAPMLVVYDDDDRPS | 124 GIPERFSGSNSGNTATLTISGVEAGDEAD | BOMA-9 Chain Vi (aa) YFCHVWDRSRDHYVFGTGTKLTVL
SYVLTQPPSVSVAPGOTARITCGGNNIER 1095 - | KNVHWYQOKPGQAPVPVVYDDSDRASG | BCMA-10 Chain V. IPERFSASNSGNTATLTISGAQATDEAEY | (aa) YCOAWDSSSTLYVFGTGTKLTVL
QLVLTQPPSASGTPGORVTISCSGSSSNI 196 - | GSNAVNWYQQLPGTAPEVLIYNSHORPS | BCMA-11 Chain V. GVPDRFSGSKSGTSASLAINGLOSEDEA | (aa) DYYCAAWDDSLRGYVFGTGTKLTVL
QSALTQPPSVSAAPGQKVTISCSGSRSNI 197 - | GNNYVSWYQQLPGTAPKLLIVDNAKRPS | BCMA-12 Chain V. GIPDRFSGSKSGTSATLDIAGLOTGDEAD! (aa) YYCQVWDSSSDHWVFGGGTKLTVL QVALVASGGGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 128 | nvrvsseceescecescecespivvt | SeFY of BOMA-1 (aa) QSPDSLSVSPGERATISCKSSQSVLSTSN NKNYLAWYQQKPGQPPRLLLYWASTRE
AGVPDRFSGSGSGTDFTLTISSLOAEDVA o SeuêNe Toei [The VeaavESSPYTFORGTRIER EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAKVDGPPSSDIWGQGTM | 129 VTVSSGGGGSGGGGSGGGGSEIVMTOS | BCMA-2 scFv (aa) PATLSVSPGETATLSCRASQSIKTNLAWY QQKPGQAPRLLIYAASTRATGIPDRFSGS GSGTDFTLTITRLEPEDFAVYYCQAQQOYGSS
PTFGRGTKLEIK QVALVASGGGLVQAPGRSLRLSCTASGPFT | FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 130 TLVTIVSSGGGGSGGGGSGGGGSDIVMT | BCMA-3 scFv, 46 (aa) QSPDSLVVSLGERATINCKSSQOSVLHSSN
NKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLOQAEDVAV! |
YYCQAQQAYYTTPLTFGGGTKVEIK QVALVASGGGLVQAQPGRSLRLSCTASGPFT | FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 131 TLVTIVSSGCGGGSGGGGSGGGGSAIRMT | BCMA-4 scFvy (aa) QSPSSLSASLGDRVTITCRASQDIRNSLA
WYQQRPGKAPKLLLSAASRLESGVPSRF SGTTSGAEYALSISSLQOPEDVASYFCQQY |
YSLPLSFGGGTKVEIK QVALVASGGGLVQAPGRSLRLSCTASGFT | FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 132 TLVTVSSGGGGSGGGGSGGGGSDIVMT | BCMA-S5 scFv (aa) QSPDSLAVSLGERATINCKSSQSVLYSSN NKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAV!
YYCAQYYSTPWTFGQGTKVDIK QVALVASGGGLVQAPGRSLRLSCTASGFT |
FGDYAMSWFRQAPGKGLEWVGFIRSKA 133 YGGTTEYAASVKGRFTISRDDSKSIAYLQ | BCMA-6 scFv (aa) MNSLKTEDTAVYYCAAWSAPTDYWGQG TLVTVSSGGGGSGGGGSGGGGSDIVMT
[4979 sean [this QSPSSLSVSVGERVTITCRASQSISNSLA WYKQRPGEAPKLLIHAASNVEDGVPSRF SGRGSGTVFTLAISNVQPEDFATYYCQQ
SHMYPPTFGGGTKVEIK QVALVASGGGLVQAPGRSLRLSCTASGFT | FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 134 TLVTIVSSGGGGSGGEGGSGGGGSVIALT | BCMA-7 scFv (aa) QSPSSLSASVGDRVTITCRASQDIGDYLA WFQQRPGKAPKSLIYVASTLOSGVPSRF SGSGSGTHFTLTINSLOPEDFATYYCQQ
YHSHPWTFGPGTKVDIK QVALVASGGGLVAPGRSLRLSCTASGFT | FGDYAMSWFRQAPGKGLEWVGFIRSKA YGGTTEYAASVKGRFTISRDDSKSIAYLQ
MNSLKTEDTAVYYCAAWSAPTDYWGQG 135 TLVTIVSSGGGGSGGGGSGGGGSDIVMT | BCMA-8 scFv (aa) QSPDSLAVSLGERATINCKSSQOSVLYSSN
NKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAV | YYCQAQQYYSTPYTFGQGTKLEIK EVOLVOAOSGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCARVDGDYVDDYWGQGT 136 LVTVSSGGGGSGGGGESGGGGSSYELTAO | BCMA-9 scFv (aa) PPSVSVAPGQOTARVTCGANNIGSKSVHW YQQKPGQAPMLVVYDDDDRPSGIPERFS GSNSGNTATLTISGVEAGDEADYFCHVW DRSRDHYVFGTGTKLTVL EVOLVESGGGLVKPGGSLRLSCAASGFP FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGPPSFDIWGQGTM 137 VTVSSGGGGSGGGGSGGGGSSYVLTOP | BCMA-10 scFv (aa) PSVSVAPGQTARITCEGGNNIERKNVHWY QQAKPGQAPVPVVYDDSDRASGIPERFSA SNSGNTATLTISGAQATDEAEYYCQAWD SSSTLYVFGTGTKLTVL QVALVASGGGLVQPGRSLRLSCAASGF SGSIGYADSVKGRF TISRDNAKNSLYLQM NSLRAEDTAVYYCARDLGPDYDPDAFDI WGOGTMVTVSSGGGEGSCEGECEGE SQLVLTQPPSASGTPGQRVTISCSGSSS NIGSNAVNWYQQLPGTAPEVLIYNSHOR PSGVPDRFSGSKSGTSASLAINGLOSED EADYYCAAWDDSLRGYVFGTGTKLTVL EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGPPSFDIWGQGTM 139 VTVSSGGEEGSCGCGGESGGGGSASALTA | BCMA-12 scFv (aa) PPSVSAAPGQKVTISCSGSRSNIGNNYVS WYQQLPGTAPKLLIVDNAKRPSGIPDRFS GSKSGTSATLDIAGLOTGDEADYYCQVW
DSSSDHWVFGGGTKLTVL BCMA-13 CDR-H1 (aa) BCMA-14 CDR-H1, -15 (aa) BCMA-16 Kabat CDR-H1 (aa) SSAMOQ BCMA-18 Kabat CDR-H1 numbering (aa) ) BCMA-19 Kabat CDR-H1 numbering (aa) SYYMH BCMA-13 Kabat CDR-H2 numbering, -14, 145 VISYDGSNKYYADSVKG -15 (aa) BCMA-16 KABAT numbering 146 WIVVGSGNTNYAQKFQE CDR-H2 (aa) ) Numbering of Kabat 147 HINQDGSEKYYVDSVKG CDR-H2 of BCMA-18 (aa) Numbering of Kabat 148 - | BCMA-19 WINPNSGGTNYAQKFOG CDR-H2 (aa) BCMA-15 Kabat Numbering DQYSSSAQRADFDY BCMA-16 (aa) APYYDILTGYYL BCMA-16 CDY-H3 (aa) EADSSADY CDR-H3 from BCMA-17 ,33
BCMA-18 ELI EEE kB WLAVTN CDR-H3 (aa) BCMA-19 DGYDV CDR-H3 (aa) 155 GGLGITPYYFDY BOMA-20 CDR-H3, 27 (aa) BCMA-21 VDGGYTEDY CDR-H3 (aa) 157 VDGDYTEDY CDMA-BC3-BC3 -23, -23, -40 (aa) BCMA-13 CDR-H1, -14, 158 GFTFSSY -15, -18 (aa) BCMA-16 Chothia CDR-H1 numbering ( aa) GFTFTSS BCMA-19 Chothia CDR-H1 numbering (aa) BCMA-13 Chothia CDR-H2 numbering, -14, 161 SYDGSN -15 (aa) BCMA-16 Chothia CDR-H2 numbering (aa) ) VVGSGN BCMA-18 Chothia CDR-H2 Numbering (aa) NODGSE BCMA-19 Chothia CDR-H2 Numbering (aa) BCMA-13 Chothia CDR-H1 Numbering (aa) GFTFSSYAMH ADM Numbering CDR-H1 BCMA-14, -15 GFTFSSYGMH (aa) BCMA-16 APM CDR-H1 numbering (aa) BCMA-18 ADM CDR-H1 numbering (aa) BCMA-19 ADM CDR-H1 numbering BCMA-19 (aa) aa) SYTFTSYYMH ADM Numbering CDR-H2 BCMA-13, -14,170 VISYDGSNKY -15 (aa) Numbering
BCMA-16 ADM CDR-H2 (aa) WIWVGSGNTN ADM numbering
BCMA-18 CDR-H2 (aa) HINQDGSEKY BCMA-19 ADM CDR-H2 (aa) BCMA-13 CDR-L1, -14, GSGSNIGSNDVS 15, -16, -18, -21 (aa) 175 | BCMA-17 coNNIGFKGVO CDR-L1, -33 (aa) BCMA-19 (aa) TGTSSDVGDYNYVA BCY-19 (aa) TGSSSDVGKYNLVS BCMA-13, -23 CDMA-13, -14, 15, 15, 16, -18, -21 (yy) 1860 | BCMA-17, -32, -33 (aa) 18635 CDR-L2 pospres - | BCMA-20 gsuDoRPS CDR-L2, -27 (aa) 163 - | BCMA-22 pvNKRPS CDR-L2, -23, -40 (aa) BCMA-13 AAWDDSLGGSWV BCMA-15 AAWDDSLSGWV BCMA-15 (aa) ASWDDSLSGWV BCMA-16 (aa) 168 - | QVywDSASDHWV BCMA-17 CDR-L3, -33 (aa) ISYSRGSTPYV BCMA-19 CDR-L3 (aa) 191 | BCMA-20, -27 (yy) GSWDDSLNAWV CDR-L3 163 | csvecsnsy BCMA-22 CDR-L3, -40 (aa) BCMA-23 SSYGGSRSYV CDR-L3 (aa) BCMA-13 QMALVAYEGEVVAPGRSLRLSCAASGF FR1 Vu (aa)
EVOLLESGGGYWAPGRSLRLSCAASGFT FR1 Va de BOMA-A (aa) 197 QVQLLESGGGLVKPGGSLRLSCAASGFT | FR1 BCMA-15, -47 FS (aa) EVALVASGPEVKKPETSVKVSCKASGFT FR1 V1 BCMA-16 (aa) 199 QVQALVASGEGLVKPGGSLRLSCAASGFT | BC1 FRx Vx-17, -33, FS -36, -38, -41 (aa)
EVOLLESGGGLVOPGGSLRLSCAASGFT QVALVASGAEVIKKPGASVIVSCKASSYT) FR1 Vu BCMA-19 (aa) EVALLESGGGLVAPERSLRLSCAASGFT FR1 Vu BCMA-20 (aa) EVOLVESGGGLVKPGGSLKLSCAASGFT FR1 Vu BCMA-21 (aa) 204 FR2 WVRQAPGKGLEWVA Vu BCMA-13, -14, -15 (aa) WVRQARGORLEWIG FR2 Vx of BCMA-16 (aa) WIRLAPGKGLEWVS FR2 Vu of BCMA-17 (aa) WHRQAPGKGPEWVA FR2 Vx of BCMA-18 (aa) WVRQAPGQGLEWMG FR2 Vy of BCMA-19 (aa) FR2 Vi: from BCMA-20, -27, WVRQAPGKGLEWVS 28, -29, -30, 34, 39 (yy) 210 RFTISRDNSKNTLYLOMNSLKAEDTAVYY | 23 y, from BCMA-13 (aa)
CAT 211 RFTISRDNSKNTLYLQMNSLRAEDTAVYY | pg3 v, from BCMA-14 (aa)
CAT 212 RFTISRDNSKNTLYLQMNSLRAEDTAVYY | ce3 v, from BCMA-15 (aa) CAK
T RVTITRDMSTSTAYMELSSLRSEDTAVYY | ce3 v, from BCMA-16 (aa)
CAA 214 RFTISRDNAESSLYLQMNSLRAEDTAVYY | 23 v, from BCMA-18 (aa)
CAR 215 RVTMTRDTSISTAYMELSRLRSDDTAVYY | cg3 v, from BCMA-19 (aa)
CAR RFTISRDNAKNSLYLQMNSLRAEDTALYY | BCMA-20 FR3 Vx, -27 216 CAK (aa) RGQGTLVTVSS BCMA-13 FR4 Vu (aa)
RGPGTLVTVSS FR4 Vu of BCMA-14 (aa) 219 WGOGTLVNVSS FR4 Vu of BCMA-17, -33 (aa) WGQGTTVTVSS FR4 Vy of BCMA-19 (aa) QAVLTQPPSASGTPGQORVTISC o. Boma V-13, -14 QSVLTAQPPSASGTPGQORVTISC Boma IN-15, -18, - 16 V. QSALTQPPSASGTPGQRVTISC BCMA-FR1 (aa) QAVLTQPASVSGSPGOSITISC FR1 V-19 BCMA (aa) .s QPVLTQPPSASGTPGQRVTISC Vi BCMA-20 -27 227 QSALTOPASVSGSPGOSITISC FR1 V. of BCMA-22, -23, - 40 (aa) FR2 V. of BCMA-13, -14, - WYQQKTGQAPVLVVY the Vi of BOMA-17, -33 WYQQHPGKDPKLMIF FR2 V. of BCMA-19 (aa) WFRQVPGTAPQLLIY o V. of BCMA-20, -27 232 WYQQPPGKAPKLIIY FR2V. BCMA-22, -23, - 40 (aa) 233 GVPDRFSASKSGTSASLAISGLRSEDEAD | 23 v, from BCMA-13 (aa) YYc 234 GVPDRFSGSKSGASASLAISGLOSEDEA | 573 y, from BCMA-14 (aa)
DYYC 235 GVPDRFSGSKSGTSASLVISGLRSEDEA | 573 y, from BCMA-15 (aa)
DYYC 236 GVPDRFSGSKSGTSASLAISGLOSEDEA | - <3 v, from BCMA-16 (aa)
DYYC GIPERFSGSNSGNTATLTISRVEAGDEAD | FR3 V. of BCMA-17, -33 237 Wc (aa) 238 GVPDRFSGSKSGTSASLAISGLRSEDEA | - <3 y, BCMA-18 (aa)
DYYC GVSDRFSGSKSGNTASLDISGLOPEDEA FR3 V. of BCMA-19 (aa)
GVPDRFSGSKSGSSASLDISGLQSEDEA | FR3 V. of BCMA-20, -27 240 YWYC (aa) 241 GVPDRFSGSKSGISASLAISGLRSEDEAD | pn3 y, from BCMA-21 (aa) ye 242 - | GVSNRFSGSKSGNTATLTISGLQGDDEA | FR3 V. of BCMA-22, -23, - DYYC 40 (aa) IGTGTKVTVL FR4 V. of BCMA-19 (aa) 245 FGGETKLTVL FR4 V. of BCMA-20, -27 (aa) 246 FGTGTKVIVL FR4 V. of BCMA -21, -22, - 23 (yy)
QMQLVAYGGGVVAPEGRSLRLSCAASGF TFSSYAMHWVRQAPGKGLEWVAVISYD: 247 GSNKYYADSVKGRFTISRDNSKNTLYLQ a Va de BOMA-IS MNSLKAEDTAVYYCATLPGRDGYPGAFD YRGQGTLVTVSS
EVOLLESGGGVVAPGRSLRLSCAASGFT FSSYGMHWVRQAPGKGLEWVAVISYDG | 248 SNKYYADSVKGRFTISRDNSKNTLYLOM BCMA-14 Va NSLRAEDTAVYYCATLPGRDGYPGAFDY RGPGTLVTVSS
QVQLLESGGGLVKPGGSLRLSCAASGFT FSSYGMHWVRQAPGKGLEWVAVISYDG: 249 SNKYYADSVKGRFTISRDNSKNTLYLQM a Va de BOMA-15 NSLRAEDTAVYYCAKDQYSSSAQRADFD YWGOGTLVTVSS
EVOLVOSGPEVKKPGTSVKVSCKASGFT FTSSAMQWVRQARGQORLEWIGWIVVGS | 250 GNTNYAQKFQERVTITRDMSTSTAYMEL BCMA-16 Va SSLRSEDTAVYYCAAAPYYDILTGYYLWG QGTLVTVSS
QVALVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRLAPGKGLEWVSYISSSGS: 251 TIYYADSVKGRFTISRDNAKNSLYLQMNS a Vade BOMAIT LRAEDTAVYYCAREADSSADYWGQGTL
VNVSS EVOLLESGGGLVQPGGSLRLSCAASGFT: 252 FSSYWMSWHRQAPGKGPEWVAHINQD to Va de BOMA-18 GSEKYYVDSVKGRFTISRDNAESSLYLQ
Fem ee
MNSLRAEDTAVYYCARWLAVTNWGQGT ss QVQLVOSGAEVKKPGASVKVSCKASGYT] FTSYYMHWVRQAPGQGLEWMGWINPN: 253 SGGTNYAQKFOGRVTMTRDTSISTAYME | BOMA-19 Chain Vu LSRLRSDDTAVYYCARDGGDVWwGQaGTT | (9th) VTVSS
EVOLLESGGGLVQPGRSLRLSCAASGFT FDDYAMHWVRQAPGKGLEWVSGISWNS: 254 GSIGYADSVKGRFTISRDNAKNsLYLamMN | BCMA-20 Chain Vu SLRAEDTALYYCAKGGLGITPYYFDYwe | (99) QGTLVTVSS
EVOLVESGGGLVKPGGSLKLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS | 255 TIYYADSVKGRFTISRDNAKNSLYLQMNS to BOMA-21 Vw LRAEDTAVYYCAKVDGGYTEDYWGQGT LVTVSS
EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS: 256 TIYYADSVKGRFTISRDNAKNSLYLQMNS Ss and aa BOMA-22, - LRAEDTAVYYCAKVDGDYTEDYWGQGT | IT'S" LVTVSS
QAVLTQPPSASGTPGQRVTISCSGSGSNI 257 - | GSNDVSWYQAIPGTAPKLLIVWNDORPS | BCMA-13 Chain V. GVPDRFSASKSGTSASLAISGLRSEDEAD | (aa) YYCAAWDDSLGGSWVFGGGTKVTVL
QAVLTQPPSASGTPGQRVTISCSGSGSNI 256 - | GSNDVSWYQAIPGTAPKLLIVWNDORPS | BCMA-14 Chain V. GVPDRFSGSKSGASASLAISGLQSEDEA | (aa) DYYCAAWDDRLNGFWVFGGGTKLTVL
QSVLTQPPSASGTPGQRVTISCSGSGSNI 256 - | GSNDVSWYQAIPGTAPKLLIVWNDORPS | BCMA-15 Chain V GVPDRFSGSKSGTSASLVISGLRSEDEA | (aa) DYYCAAWDDSLSGWVFGGGTKLTVL
QSALTQPPSASGTPGQRVTISCSGSGSNI 260 - | GSNDVSWYQQIPGTAPKLLIVWNDORPS | BCMA-16 Chain V. GVPDRFSGSKSGTSASLAISGLOSEDEA | (aa)
DYYCASWDDSLSGWVFGGGTKLTVL QPVLTQPPSVSVAPGKTAMITCGGNNIGF | IPERFSGSNSGNTATLTISRVEAGDEADY
[REP [ssa Joss = | | | yYeawosASsDAWVEGGETREIVE - | QSVLTQPPSASGTPGQRVTISCSGSGSN! I 262 GSNDVSWYQQIPGTAPKLLIYWNDORPS | BCMA-18 Chain V. GVPDRFSGSKSGTSASLAISGLRSEDEA | (aa) DYYCAAWDDSLNGWVFGGGTKLTVL
QAVLTQPASVSGSPGOSITISCTGTSSDV 263 GDYNYVAWYQQHPGKDPKLMIFEVINRP | BCMA-19 Chain V. SGVSDRFSGSKSGNTASLDISGLQPEDE | (aa) ADYYCISYSRGSTPYVIGTGTKVTVL
QPVLTQPPSASGTPGQRVTISCSGGKTV 264 NWFRQVPGTAPQLLIYSNDORPSGVPDR | Chain V, BCMA-20, - FSGSKSGSSASLDISGLQOSEDEAYYYCG | 27 (aa) SWDDSLNAWVFGGETKLTVL
QSVLTQPPSASGTPGQRVTISCSGSGSNI 265 GSNDVSWYQQIPGTAPKLLIYWNDORPS | BCMA-21 Chain V. GVPDRFSGSKSGISASLAISGLRSEDEAD | (aa) YYCAAWDDSLNGYVFGTGTKVTVL
QSALTQPASVSGSPGOSITISCTGSSSDV 266 GKYNLVSWYQQPPGKAPKLIIYDVNKRP | BCMA-22 Chain V. SGVSNRFSGSKSGNTATLTISGLQGDDE | (aa) ADYYCCSYGGSRSYVFGTGTKVTVL
QSALTQPASVSGSPGOSITISCTGSSSDV 267 GKYNLVSWYQQPPGKAPKLIIYDVNKRP | BCMA-23 Chain V. SGVSNRFSGSKSGNTATLTISGLOGDDE | (aa) ADYYCSSYGGSRSYVFGTGTKVTVL QMQLVAQYGGGVVQPGRSLRLSCAASGF TFSSYAMHWVRQAPGKGLEWVAVISYD GSNKYYADSVKGRFTISRDNSKNTLYLQ
MNSLKAEDTAVYYCATLPGRDGYPGAFD 268 YRGQGTLVTIVSSGGGGSGGGGSGGGG | BCMA-13 scFv (aa) SQAVLTAQPPSASGTPGQRVTISCSGSGS NIGSNDVSWYQQIPGTAPKLLIYWNDOR PSGVPDRFSASKSGTSASLAISGLRSEDE ADYYCAAWDDSLGGSWVFGGGTKVTVL EVOLLESGGGVVOQPGRSLRLSCAASGFT FSSYGMHWVRQAPGKGLEWVAVISYDG
SNKYYADSVKGRFTISRDNSKNTLYLQM 269 NSLRAEDTAVYYCATLPGRDGYPGAFDY | BCMA-14 scFv (aa) RGPGTLVTVSSGGGGSCGGGSCGEGS QAVLTQPPSASGTPGQRVTISCSGSGSN! I GSNDVSWYQQIPGTAPKLLIYWNDORPS
[57 | semes oesgiving
GVPDRFSGSKSGASASLAISGLQSEDEA | wemmenRaMeÊvGBTA QVQLLESGGGLVKPGGSLRLSCAASGFT FSSYGMHWVRQAPGKGLEWVAVISYDG SNKYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCAKDOYSSSAQRADFD 270 | YWGOGTLVTIVSSEGGGSGGGESGGGG | BCMA-15 scFv (aa) SQSVLTAPPSASGTPGQRVTISCSGSGS NIGSNDVSWYQQIPGTAPKLLIVWNDOR PSGVPDRFSGSKSGTSASLVISGLRSED EADYYCAAWDDSLSGWVFGGGTKLTVL EVOLVOSGPEVKKPGTSVKVSCKASGFT FTSSAMOWVRQARGORLEWIGWIVVGS GNTNYAQKFQERVTITRDMSTSTAYMEL
SSLRSEDTAVYYCAAAPYYDILTGYYLWG 271 QGTLVTIVSSGGGESCGEGESGGGESAS | BCMA-16 scFv (aa) ALTQPPSASGTPGQRVTISCSGSGSNIGS | NDVSWYQQIPGTAPKLLIVWNDORPSGV PDRFSGSKSGTSASLAISGLOSEDEADY YCASWDDSLSGWVFGGGTKLTVL QVALVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRLAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAREADSSADYWGQGTL 272 - | VNVSSGGGGSGEGESGEGGESAPVLTA | BCMA-17 scFv (aa) PPSVSVAPGKTAMITCGGNNIGFKGVQW YQQKTGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVW DSASDHWVFGGGTKLTVL EVOLLESGGGLVQPGGSLRLSCAASGFT FSSYWMSWHRQAPGKGPEWVAHINQD GSEKYYVDSVKGRFTISRDNAESSLYLQ
MNSLRAEDTAVYYCARWLAVTNWGQOGT 273 | LVTIVSSEGGGSGEGESCGEGESASVLTA | BCMA-18 scFv (aa) PPSASGTPGQRVTISCSGSGSNIGSNDV
SWYQQIPGTAPKLLIVWNDQRPSGVPDR FSGSKSGTSASLAISGLRSEDEADYYCAA | WDDSLNGWVFGGGTKLTVL QVQLVOSGAEVKKPGASVKVSCKASGYT
FTSYYMHWVRQAPGQGLEWMGWINPN 274 SGGTNYAQKFOGRVTMTRDTSISTAYME | BCMA-19 SvFvY (aa) LSRLRSDDTAVYYCARDGGDVWGOGTT
[RPE if that VTVSSGGGGSCGGGGSCGGGSAAVLTA PASVSGSPGOSITISCTGTSSDVGDYNYV) AWYQQHPGKDPKLMIFEVINRPSGVSDR FSGSKSGNTASLDISGLQPEDEADYYCIS YSRGSTPYVIGTGTKVTVL EVOLLESGGGLVQPGRSLRLSCAASGFT FDDYAMHWVRQAPGKGLEWVSGISWNS GSIGYADSVKGRFTISRDNAKNSLYLQMN
SLRAEDTALYYCAKGGLGITPYYFDYWG 275 QGTLVYTVSSEGGGSGGGGSGGGGSQAP | BCMA-20 scFv (aa) VLTQPPSASGTPGQRVTISCSGGKTVNW FRQVPGTAPOQLLIYSNDORPSGVPDRFS GSKSGSSASLDISGLOSEDEAYYYCGSW DDSLNAWVFGGETKLTVL EVOLVESGGGLVKPGGSLKLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGGYTEDYWGQOGT 276 LVTVSSGGGGSGGGESGGGGSASVLTA | BCMA-21 scFv (aa) PPSASGTPGQRVTISCSGSGSNIGSNDV SWYQQIPGTAPKLLIYWNDORPSGVPDR FSGSKSGISASLAISGLRSEDEADYYCAA WDDSLNGYVFGTGTKVTVL EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGDYTEDYWGQGT 277 LVTVSSGGGGSGGGGSGGGGSASALTAO | BCMA-22 scFv (aa) PASVSGSPGOQSITISCTGSSSDVGKYNLV SWYQQPPGKAPKLIIYDVNKRPSGVSNR FSGSKSGNTATLTISGLOGDDEADYYCC SYGGSRSYVFGTGTKVTVL EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGDYTEDYWGQGT 278 LVTVSSGGGGSGGGGSGGGGSASALTAO | BCMA-23 scFv (aa) PASVSGSPGOQSITISCTGSSSDVGKYNLV SWYQQPPGKAPKLIIYDVNKRPSGVSNR FSGSKSGNTATLTISGLOGDDEADYYCS SYGGSRSYVFGTGTKVTVL
DPLSWDSSGKGPR CDR-H3 V-4 (aa) ENYDFWSWRYYYDMDV CDR-H3 Vr-5 (aa) VDGPPSYDI CDR-H3 Vr-6 (aa) GDWDDAFDI CDR-H3 Vr-7 (aa) VDGDYEDY CDR-H3 (aa) VDGDYEDY CDR-H3 DVPSSGDDAFDI CDR-H3 V.-10 (aa) VDGDDVFDI CDR-H3 Vr-11 (aa) VDGDAFDI CDR-H3 Vr-12 (aa) 288 DYSIN CDR-H1 Vu from BCMA-C1 (aa) 289 NFGMN CDR-H1 Vu from BCMA-C2 (aa) WINTETREPAYAYDFRG to Va from BOMA-D1 WINTYTGESYFADDFKG to Va from BCMA-C2 292 DYSYAMDY CDR-H3 Vu from BCMA-C1 (aa) GEIYYGYDGGFAY CDR-H3 Vu BCMA-C2 BCMA-C1 Vu 294 GYTFTDY (aa) Chothia CDR-H1 Vu Numbering BCMA-C2 295 GYTFTNF (aa) Chothia CDR-H2 Vu Numbering BCMA-C1 296 NTETRE (aa) Chothia Numbering CDR-H2 Vu de BCMA-C2 297 NTYTGE (aa) BCMA-C1 SYTFTDYSIN Chothia CDR-H1 Vu numbering (aa) BCMA-C2 GYTFTNFGMN ADM CDR-H1 Vu numbering (aa) BCMA-C1 WINTETREPAPA APM CDR-H2 Vu numbering (aa) Numbering of ADM CDR-H2 Vu of BCMA-C2 WINTYTGESY (aa) Numbering of APM
RASESVTILGSHLIH to V. of BCMA-C1 303 RASQDVNTAVS CDR-L1 V. of BCMA-C2 (aa) 304 LASNVOT CDR-L2 V. of BCMA-C1 (aa) 305 SASYRYT CDR-L2 V. of BCMA-C2 (aa) LOSRTIPRT 306 CDR-L3 V. BCMA-C1 (aa) CDR-L3 QQHYSTPWT 307 V. BCMA-C2 (aa) QIALVASGPELIKKPEETVKISCKASGYTF FR1 Va BCMA-C1 (aa) GIALVASSPDLIKPOETVKLSCKASGYTF FR1 V BCMA-C2 (aa) RFAFSLETSASTAVLOINNLKYEDTATYF FR3 Vi of BCMA-C1 (aa) 313 RFAFSVETSATTAYLQINNLKTEDTATYF | c23 y, from BCMA-C2 (aa)
CAR WGQGTSVTVSS FR4 BCMA-C1 Vu (aa) WGQGTLVTVSA FR4 BCMA-C2 Vu (aa) DIVLTOSPPSLAMSLGKRATISC FR1 V. of BCMA-C1 (aa) DVVMTOSHRFMSTSVGDRVSITC FRQ V.QY-2 C1 (aa) WYQQKPGQOSPKLLIF FR2 V. of BCMA-C2 (aa) 320 GVPARFSGSGSRTDFTLTIDPVEEDDVAV | pr3 v, BCMA-C1 (aa) YYc 321 GVPDRFTGSGSGADFTLTISSVQAEDLAV | pg3 v, BCMA-C2 (aa) YYc
QIQLVOSGPELKKPGETVKISCKASGYTF TDYSINWVKRAPGKGLKWMGWINTETRE: 324 PAYAYDFRGRFAFSLETSASTAYLQINNL ão Va de BOMA-C1 KYEDTATYFCALDYSYAMDYWGQOGTSV TVSS
Fes and
QIQLVASGPDLKKPGETVKLSCKASGYTF TNFGMNWVKQAPGKGFKWMAWINTYTG | 325 ESYFADDFKGRFAFSVETSATTAYLQINN | BCMA-C2 Vu Chain LKTEDTATYFCARGEIYYGYDGGFAYWwG | (92) QGTLVTVSA
DIVLTAQSPPSLAMSLGKRATISCRASESV 326 - | TILGSHLIHWYQOKPGQPPTLLIQLASNV | BCMA-C1 Chain V. QTGVPARFSGSGSRTDFTLTIDPVEEDD | (aa) VAVYYCLOSRTIPRTFGGGTKLEIK
DVVMTOSHRFMSTSVGDRVSITCRASQD 327 - | VNTAVSWYQOKPGOSPKLLIFSASYRYT | BCMA-C2 Chain V. GVPDRFTGSGSGADFTLTISSVQAEDLAV | (aa) YYCAQHYSTPWTFGGGTKLDIK DIVLTASPPSLAMSLGKRATISCRASESV TILGSHLIHWYQQKPGQAPPTLLIQLASNV QTGVPARFSGSGSRTDFTLTIDPVEEDD
VAVYYCLOSRTIPRTFGGGTKLEIKGGGG 328 SEGGEGESCGEGESAIALVASGPELKKPGE a) VeVade BOMA-O1 TVKISCKASGYTFTDYSINWVKRAPGKGL KWMGWINTETREPAYAYDFRGRFAFSLE TSASTAYLQINNLKYEDTATYFCALDYSY AMDYWGQGTSVTVSS QIQLVASGPDLKKPGETVKLSCKASGYTF TNFGMNWVKQAPGKGFKWMAWINTYTG ESYFADDFKGRFAFSVETSATTAYLQINN
LKTEDTATYFCARGEIYYGYDGGFAYWG 329 QGTLVIVSAGGGESGEGESGEGESDV | SeFY VV. BCMA-C2 VMTOSHRFMSTSVGDRVSITCRASQDVN | (92) TAVSWYQQKPGQSPKLLIFSASYRYTGV PDRFTGSGSGADFTLTISSVQAEDLAVYY CQQHYSTPWTFGGGTKLDIK CAGGTGCAGCTGGTGCAGTCTGGGEG AGGCTTGGTACAGCCAGGGCEGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG
GTTCCGCCAGGCTCCAGGGAAGGGGC 330 - | TGGAGTGGGTAGGTTTCATTAGAAGCA | BCMA-1 scFv (nt) AAGCTTATGGTGGGACAACAGAATACG CCGCEGTCTGTGAMAGGCAGATTCACCA TCTCAAGAGATGATTCCAAMAGCATCG CCTATCTGCAAATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG
[4979 sean [this CGGCCTGGAGTGCCCCGACTGACTACT GGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGGTGGAGGCGGTTCAGGCEGG AGGTGGCTCTGGCGGTEGCGGATCGG ATATTGTGATGACCCAGTCTCCAGACTC, CCTGTCTGTGTCTCCGGGCGAGAGGEG CCACCATCAGCTGCAAGTCCAGCCAGA GTGTTTTATCCACCTCCAACAATAAGAA CTATTTAGCTTGGTATCAGCAGAAACCA GGACAGCCCCCTAGGCTGCTCCTTTAC
TGGGCATCTACCCGGGAGGCCGGEGT CCCTGACCGATTCAGTGGCAGCGGGTC | TGGGACAGATTTCACTCTCACCATCAG CAGCCTGCAGGCTGAAGATGTGGCGG TTTATTACTGTCAACAATATTTCAGTTCT CCGTACACTTTTGGCCACGGGACCAAG CTGGAAATCAAA GAGGTGCAGCTGGTGGAGTCTGGGGGÇ AGGCTTGGTCAAGCCTGGAGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTGACTACTACATGAGCTG GATCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGG GACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAAAGTG
GATGGCCCTCCTTCTTCTGATATCTGG 331 GGCCAAGGGACAATGGTCACCGTCTCC | BCMA-2 scFvy (nt) TCAGGTGGAGGCGGTTCAGGCGGAGG TGGCTCTGGCGGTGGCEGGATCGGAAA TAGTGATGACGCAGTCTCCAGCCACCC TGTCTGTGTCTCCAGGGGAAACAGCCA CCCTCTCCTGCAGEGCCAGTCAGAGTA TTAAGACCAACTTGGCCTGGTACCAGC AGAAACCTGGCCAGGCTCCCAGGCTCC TCATCTATGCTGCATCCACCAGGGCCA CTGGCATCCCAGACAGATTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCA CCATCACCAGACTGGAGCCTGAAGATT TTGCAGTGTATTACTGTCAGCAATATGG
[57 | seeuênea The eesemão
TAGCTCACCCACTTTTGGCCOGECÇAC | EMeeTGBAATEAA CAGGTGCAGCTGGTGCAGTCTGGGEG AGGCTTGGTACAGCCAGGGCGGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTAGGTTTCATTAGAAGCA AAGCTTATGGTGGGACAACAGAATACG CCGCEGTCTGTGAMAGGCAGATTCACCA TCTCAAGAGATGATTCCAAMAGCATCG CCTATCTGCAAMATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CEGGCCTGGAGTGCCCCGACTGACTACT GGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAGGTGGAGGCGGTTCAGGCGG 332 AGGTGGCTCTGGCEGTEGCEGATCEÇ | BOMA-3 SCFY (nt) ATATTGTGATGACCCAGTCTCCAGACTCI CCTGGTTGTGTCTCTGGGCGAGAGEG CCACCATCAACTGCAAGTCCAGCCAGA GTGTTTTACACAGCTCCAACAATAAGAA TTACTTAGCTTGGTACCAGCAGAAACCA GGACAGCCTCCTAAGCTGCTCATTTAC TGGGCATCTACCCGGGAATCCGGGGT CCCTGACCGGTTCAGTGGCAGCGGGT CTGGGACAGATTTCACTCTCACCATCA GCAGCCTGCAGGCTGAAGATGTGGCA GTTTATTACTGTCAGCAGTATTATACTA CTCCGCTCACTTTCGGCEGAGGGACCA AGGTGGAAATCAAA CAGGTGCAGCTGGTGCAGTCTGGGGGE AGGCTTGGTACAGCCAGGGCGGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGEGGEC
TGGAGTGGGTAGGTTTCATTAGAAGCA 333 AAGCTTATGGTGGGACAACAGAATACG | BOMA-4 SCFV (nt) CCGCEGTCTGTGAMAGGCAGATTCACCA TCTCAAGAGATGATTCCAAAAGCATCG CCTATCTGCAAMATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CEGGCCTGGAGTGCCCCGACTGACTACT
[4979 sean [this GGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGGTGGAGGCGGTTCAGGCGEG AGGTGGCTCTGGCGGTEGGCGGATCGG CCATCCGGATGACCCAGTCTCCATCCOT CCCTEGTCCGCGTCTCTGGGEGACAGA GTCACCATCACTTGCCGGGCGAGTCAG GACATTAGGAATTCTTTGGCCTGGTATC AGCAGAGGCCAGGGAAAGCCCCTAAA CTCCTGCTTTCTGCTGCATCCAGATTG GAAAGTGGGGTCCCTTCTAGGTTCAGT GGCACTACTTCTGGGGCGGAGTATGCT CTCAGCATCAGCAGCCTGCAGCCTGAA GATGTCGCATCTTATTTCTGTCAGCAGT ATTATAGTCTCCCTCTCTCCTTCGGCGG AGGGACCAAGGTGGAAATCAAA CAGGTGCAGCTGGTGCAGTCTGGGGG AGGCTTGGTACAGCCAGGGCGGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTAGGTTTCATTAGAAGCA AAGCTTATGGTGGGACAACAGAATACG CCGCETCTGTGAAAGGCAGATTCACCA TCTCAAGAGATGATTCCAAAAGCATCG CCTATCTGCAAATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CGGCCTGGAGTGCCCCGACTGACTACT
GGGGCCAGGGAACCCTGGTCACCGTC 334 TCCTCAGGTGGAGGCGGTTCAGGCGG | BCMA-S scFy (nt) AGGTGGCTCTGGCGGTEGCGGATCGG ACATCGTGATGACCCAGTCTCCAGACT CCCTGGCTGTGTCTCTGGGCGAGAGG GCCACCATCAACTGCAAGTCCAGCCAG AGTGTTTTATACAGCTCCAACAATAAGA ACTACTTAGCTTGGTACCAGCAGAAAC CAGGACAGCCTCCTAAGCTGCTCATTT ACTGGGCATCTACCCGGGAATCCGGG GTCCCTGACCGATTCAGTGGCAGCGG GTCTGGGACAGATTTCACTCTCACCAT CAGCAGCCTGCAGGCTGAAGATGTGG CAGTTTATTACTGTCAGCAATATTATAG TACTCCGTGGACGTTCGGCCAAGGGAC
[67 | seuenea o eee | | CcAAGGTGGATATEMRMA | CAGGTGCAGCTGGTGCAGTCTGGGGEG AGGCTTGGTACAGCCAGGGCEGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTAGGTTTCATTAGAAGCA AAGCTTATGGTGGGACAACAGAATACG CCGCETCTGTGAMAGGCAGATTCACCA TCTCAAGAGATGATTCCAAMAGCATCG CCTATCTGCAAMATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CGGCCTGGAGTGCCCCGACTGACTACT
GGGGCCAGGGAACCCTGGTCACCGTC 335 | TCCTCAGGTGGAGGCGGTTCAGGCGG | BCMA-6 scFv (nt) AGGTGGCTCTGGCGGTGGECGGATCGG ATATTGTGATGACCCAGTCTCCATCGTC CCTGTCTGTGTCTGTAGGAGAGAGAGT CACCATCACTTGTCGGGCGAGTCAGTC TATAAGTAATTCCTTAGCCTGGTATAAA CAGAGACCGGGAGAAGCCCCTAAACTC CTGATACATGCTGCATCCAATGTGGAA GATGGGGTCCCTTCGAGGTTCAGCGG CAGGGGATCTGGGACAGTTTTCACTCT CGCCATCAGCAATGTACAGCCTGAAGA TTTCGCAACTTACTACTGTCAGCAGAGT CACATGTACCCTCCGACTTTCGGCEGG GGGACCAAGGTGGAAATCAAA CAGGTGCAGCTGGTGCAGTCTGGGGEG AGGCTTGGTACAGCCAGGGCEGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGEGEC TGGAGTGGGTAGGTTTCATTAGAAGCA
AAGCTTATGGTGGGACAACAGAATACG 336 CCECETCTGTGAMAGGCAGATTCACCA | SF of BOMA-7 (nt) TCTCAAGAGATGATTCCAAMAGCATCG CCTATCTGCAAMATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CGGCCTGGAGTGCCCCGACTGACTACT GGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGGTGGAGGCGGTTCAGECEG
[57 | seeuênea the same month AGGTGGCTCTGCCECTCECEGATCGGE TCATCCAGTTGACCCAGTCTCCCTCCT CACTGTCTGCATCTGTAGGGGACAGAG TCACCATCACTTGTCGGGCGAGTCAGG ACATTGGCGATTATTTAGCCTGGTTTCA GCAGAGACCAGGGAAAGCCCCTAAGTC
CCTGATCTATGTTGCGTCCACTTTGCAG AGTGGGGTCCCATCAAGGTTCAGCGGC | AGTGGATCTGGGACACACTTCACTCTC ACCATCAACAGCCTGCAGCCTGAAGAT TTTGCAACTTATTACTGCCAACAGTATC ATAGTCACCCGTGGACGTTCGGCCCAG GGACCAAGGTGGATATCAAA CAGGTCCAGCTGGTGCAGTCTGGEGG AGGCTTGGTACAGCCAGGGCEGTCCC TGAGACTCTCCTGTACAGCTTCTGGATT CACCTTTGGTGATTATGCTATGAGCTG GTTCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTAGGTTTCATTAGAAGCA AAGCTTATGGTGGGACAACAGAATACG CCGCETCTGTGAMAGGCAGATTCACCA TCTCAAGAGATGATTCCAAMAGCATCG CCTATCTGCAAMATGAACAGCCTGAAAA CCGAGGACACAGCCGTGTATTACTGTG CGGCCTGGAGTGCCCCGACTGACTACT GGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAGGTGGAGGCGGTTCAGECEG 37 AGGTGGCTCTGGCGGTEGCEGATCEÇ | BOMA-8 SCFY (nt) ATATTGTGATGACCCAGTCTCCAGACTCI CCTGGCTGTEGTCTCTGEGCGAGAGEG CCACCATCAACTGCAAGTCCAGCCAGA GTGTTTATACAGCTCCAACAATAAGAA CTACTTAGCTTGGTACCAGCAGAAACC AGGACAGCCTCCTAAGCTGCTCATTTA CTGGGCATCTACCCGGGAATCCGGGG TCCCTGACCGATTCAGTGGCAGCGGGT CTGGGACAGATTTCACTCTCACCATCA GCAGCCTGCAGGCTGAAGATGTGGCA GTTTATTACTGTCAGCAATATTATAGTA CTCCGTACACTTTTGGCCAGGGGACCA AGCTGGAAATCAAA
[57 | seeuênea The “mesenão AGGCTTGGTCAAGCCTGGAGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTGACTACTACATGAGCTG GATCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGG GACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGTG GACGGTGACTACGTCGATGACTACTGG GGCCAGGGAACCCTGGTCACCGTCTC CTCAGGTGGAGGCGGTTCAGGCGGAG GTGGCTCTGGCGGTGGCGGATCGTCC TATGAGCTGACTCAGCCGCCCTCGGTG TCTGTGGCCCCAGGACAGACGGCCAG GGTTACCTGTGGGGCAAATAATATTGG AAGCAAAAGTGTCCACTGGTACCAGCA GAAGCCAGGCCAGGCCCCCATGCTEG TCGTCTATGATGATGACGACCGGCCCT CCGGGATCCCTGAGCGATTCTCTGGCT CCAACTCTGGGAACACGGCCACCCTGA CCATCAGCGGGGTCGAGECCGGEGAT GAGGCCGACTACTTCTGTCACGTGTGG GATAGAAGTCGTGATCATTATGTCTTCG GAACTGGGACCAAGCTGACCGTCCTA GAAGTGCAGCTGGTGCAGTCTGGGEGG AGGCTTGGTCAAGCCTGGAGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTGACTACTACATGAGCTG GATCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT
GTGAAGGGCCGATTCACCATCTCCAGG 339 GACAACGCCAAGAACTCACTGTATCTG | BCMA-10 SCFvY (nt) CAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGTG GACGGTGACTACGTCGATGACTACTGG GGCCAGGGAACCCTGGTCACCGTCTC CTCAGGTGGAGGCGGTTCAGGCGGAG GTGGCTCTGGCGGTEGCGGATCGTCC TATGAGCTGACTCAGCCGCCCTCGGTGE
[57 | seeuênea eesenão TCTGTGGCCCCAGGACAGACGGCCAG GGTTACCTGTGGGGCAAATAATATTGG AAGCAAAAGTGTCCACTGGTACCAGCA GAAGCCAGGCCAGGCCCCCATGCTGG TCGTCTATGATGATGACGACCGGCCCT CCGGGATCCCTGAGCGATTCTCTGGCT CCAACTCTGGGAACACGGCCACCCTGA CCATCAGCGGGGTCGAGGCCGGEGGAT GAGGCCGACTACTTCTGTCACGTGTGG GATAGAAGTCGTGATCATTATGTCTTCG GAACTGGGACCAAGCTGACCGTCCTA CAGGTGCAGCTGGTACAGTCTGGGEGGE AGGCTTGGTACAGCCTGGCAGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTTGATGATTATGCCATGCACTGG GTCCGGCGAGCTCCAGGGAAGGGCCT GGAGTGGGTCTCAGGTATTAGTTGGAA TAGTGGTAGCATAGGCTATGCGGACTC TGTGAAGGGCCGATTCACCATCTCCAG AGACAACGCCAAGAACTCCCTGTATCT GCAAATGAACAGTCTGAGAGCTGAGGA CACGGCCGTGTATTACTGTGCGAGAGA TCTGGGGCCCGACTACGATCCCGATGC TTTTGATATCTGGGGCCAAGGGACAAT
GGTCACCGTTTCCTCAGGTGGAGGCG 3rd | grrcacececaceteccTCTGGCEET | SFY of BOMA-11 (nt) GGCGGATCGCAGCTTGTGCTGACTCAG CCACCCTCAGCGTCTGGGACCCCCGG GCAGAGGGTCACCATCTCTTGTTCTGG AAGCAGCTCCAACATCGGAAGTAATGC TGTAAACTGGTACCAGCAGCTCCCAGG AACGGCCCCCGAAGTCCTCATCTATAA TAGTCATCAGCGGCCCTCAGGGGTCCC TGACCGATTCTCTGGCTCCAAGTCTGG CACCTCAGCCTCCCTGGCCATCAATGG GCTCCAGTCTGAGGACGAGGCTGATTA TTACTGTGCAGCATGGGATGACAGCCT GAGAGGTTACGTCTTCGGAACTGGGAC CAAGCTCACCGTCCTA
GAGGTGCAGCTGGTGGAGTCTGGGGGE 341 - | AGGCTTGGTCAAGCCTGGAGGGTCCCT | BCMA-12 scFv (nt) GAGACTCTCCTGTGCAGCCTCTGGATT
[57 | seeuênea The Aeseão CACCTTCAGTGACTACTACATGAGCTG GATCCGCCAGGCTCCAGEGAAGGEGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGG GACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAAAGTG GATGGCCCTCCTTCTITTGATATCTGGG GCCAAGGGACAATGGTCACCGTCTCCT CAGGTGGAGGCGGTTCAGECGGAGGT GGCTCTGGCGEGTGGCGGATCGCAGTC TGCCCTGACGCAGCCGCCCTCAGTGTC TGCGGCCCCAGGACAGAAGGTCACCAT CTCCTGCTCTGGAAGCCGCTCCAACAT TGGGAATAATTATGTATCCTGGTACCAA CAGCTCCCAGGAACAGCCCCCAAACTC CTCATTTATGACAATGCTAAGCGACCCT CAGGAATTCCTGACCGATTCTCTGGCT CCAAGTCTGGCACGTCAGCCACCCTGG ACATCGCCGGACTCCAGACTGGGGATG AGGCCGACTATTACTGTCAGGTGTGGG ATAGTAGTAGTGATCATTGGGTATTCGG CGGAGGGACCAAGCTCACCGTCCTA CAGATGCAGCTGGTGCAGTATGGGGGE AGGCGTGGTCCAGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGAT TCACCTTCAGTAGCTATGCTATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGEC TGGAGTGGGTGGCAGTTATATCATATG ATGGAAGTAATAAATACTACGCAGACTC CGTGAAGGGCCGATTCACCATCTCCAG
AGACAATTCCAAGAACACGCTGTATCT 342 GCAAATGAACAGCCTGAAAGCTGAGGA | BOMA-13 SCFv (nt) CACGGCTGTGTATTACTGTGCTACCCT ACCCGGTAGAGATGGCTACCCCGGAG CCTTTGACTACAGGGGCCAGGGAACCC TGGTCACCGTCTCCTCAGGTGGAGGCG GTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGCAGGCTGTGCTGACTCA GCCACCCTCAGCGTCTGGGACCCCCG GGCAGAGGGTCACCATCTCTTGTTCTG
[57 | SeBBEREA oesemão GAAGCGGCTCCAACATCGGAAGTAATG ATGTCTCCTGGTATCAGCAGATCCCAG GAACGGCCCCCAAACTCCTCATCTACT GGAATGATCAGCGGCCCTCAGGGGTC CCTGACCGATTCTCTGCCTCCAAGTCT GGCACCTCAGCCTCCCTGGCCATCAGT GGGCTCCGETCCGAGGATGAGGCTGA TTATTACTGTGCAGCATGGGATGACAG CCTGGGTGGETTCTTGGETETTCGGCGG AGGGACCAAGGTCACCGTCCTA GAGGTGCAGCTGTTGGAGTCTGGEGG AGGCGTGGTCCAGCCTGGGAGGTCCC TGAGACTCTCCTGTGCAGCGTCTGGAT TCACCTTCAGTAGCTATGGCATGCACT GGEGTCCGCCAGGCTCCAGGCAAGEGG CTGGAGTGGGTGGCAGTTATATCATAT GATGGAAGTAATAAATACTACGCAGACT CCEGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCTGAGG ACACGGCTGTGTATTACTGTGCTACCC TACCCGGTAGAGATGGCTACCCCGGAG CCTTTGACTACAGGGGCCCGEGAACCC
TGGTCACCGTCTCCTCAGGTGGAGGCG 33 GTTCAGGCGGAGGTGGCTCTGGCGEGT | SCFY of BOMA-14 (nt) GGCGGATCGCAGGCTGTGCTGACTCA GCCACCCTCAGCGTCTGGGACCCCCG GGCAGAGGGTCACCATCTCTTGTTCTG GAAGCGGCTCCAACATCGGAAGTAATG ATGTCTCCTGGTATCAGCAGATCCCAG GAACGGCCCCCAAACTCCTCATCTACT GGAATGATCAGCGGCCCTCAGGGGTC CCTGACCGATTCTCTGGCTCCAAGTCT GGCGCCTCAGCCTCTCTGGCCATCAGT GGGCTCCAGTCTGAGGATGAGGCTGAT TATTATTGTGCAGCATGGGATGACAGG TTGAACGGTTTTTGGGTGTTCGGCGGA GGGACCAAGCTCACCGTCCTA CAGGTGCAGCTGTTGGAGTCTGGGEG
AGGCCTGGTCAAGCCTGGGGGETCCC At TGAGACTCTCCTGTGCAGCCTCTGGAT | SeFv of BOMA-15 (nt) TCACCTTCAGTAGCTATGGCATGCACT
[5 | SEBBEREA oesemão GCECTCCGSCCAGGCTCCAGGCAAGEGG CTGGAGTGGGTGGCAGTTATATCATAT GATGGAAGTAATAAATACTATGCAGACT CCGTGAAGGGCCGATTCACCATCTCCA GAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCTGAGG ACACGGCTGTGTATTACTGTGCGAAAG ATCAGTATAGCAGTAGCGCACAAAGGG CCGACTTTGACTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGGTGGAG GCGGTTCAGGCGGAGGTGGTTCTGGC GGTGGCGGATCGCAGTCTGTGCTGAC GCAGCCACCCTCAGCGTCTGGGACCC CCGGGCAGAGGGTCACCATCTCTTGTT CTGGAAGCGGCTCCAACATCGGAAGTA ATGATGTCTCCTGGTATCAGCAGATCC CAGGAACGGCCCCCAAMACTCCTCATCT ACTGGAATGATCAGCGGCCCTCAGGG GTCCCTGACCGGTTCTCAGGCTCCAAG TCTGGCACCTCAGCCTCCCTGGTCATC AGTGGGCTCCGGTCCGAGGATGAGGC TGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTGGTTGGGTGTTCGGCG GAGGGACCAAGCTGACCGTCCTA GAGGTCCAGCTGGTACAGTCTGGGCCT GAGGTGAAGAAGCCTGGGACCTCAGT GAAGGTCTCCTGCAAGGCTTCTGGATT CACCTTTACTAGCTCTGCTATGCAGTG GGTGCGACAGGCTCGTGGACAACGCC TTGAGTGGATAGGATGGATCGTCGTTG GCAGTGGTAACACAAACTACGCACAGA AGTTCCAGGAAAGAGTCACCATTACCA
GGGACATGTCCACAAGCACAGCCTACA TGGAGCTGAGCAGCCTGAGATCCGAG | SeFv of BOMA-16 (nt) GACACGGCCGTGTATTACTGTGCGGCA GCTCCGTATTACGATATTTTGACTGGTT ATTATTTATGGGGCCAGGGAACGCTGG TCACCGTCTCCTCAGGTGGAGGCEGTT CTGGCGGAGGTGGCTCTGGCGGTEGC GGATCGCAGTCTGCCCTGACTCAGCCA CCCTCAGCGTCTGGGACCCCCGEGCA GAGGGTCACCATCTCTTGTTCTGGAAG
[4979 sean [this CGGCTCCAACATCGGAAGTAATGATGT CTCCTGGTATCAGCAGATCCCAGGAAC
GGCCCCCAAACTCCTCATCTACTGGAA TGATCAGCGGCCCTCAGGGGTCCCTGA | CCGATTCTCTGGCTCCAAGTCTGGCAC CTCAGCCTCCCTGGCCATCAGTGGGCT CCAGTCTGAGGATGAGGCTGATTATTA CTGTGCATCATGGGATGACAGCCTGAG TGGTTGGGTGTTCGGCGGAGGGACCA AGCTGACCGTCCTA
CAGGTTCAGCTGGTGCAGTCTGGGGG AGGCTTGGTCAAGCCTGGAGGGTCCCT | GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTGACTACTACATGAGCTG GATCCGCCTGGCTCCAGEGAAGGGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGG GACAACGCCAAGAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGAC
ACGGCCGTGTATTACTGTGCGAGAGAG GCCGATAGTAGCGCTGACTACTGGGGC |
CAGGGAACCCTGGTCAACGTCTCCTCA 346 GGTGGAGGCGGTTCAGGCGGAGGTGG | BCMA-17 scFvy (nt) CTCTGGCGGTGGCGGATCGCAGCCTG TGCTGACTCAGCCACCCTCGGTGTCAG TGGCCCCAGGAAAGACGGCCATGATTA CCTGTGGGGGAAACAACATTGGATTTA
AAGGTGTGCAGTGGTACCAGCAGAAGA CAGGCCAGGCCCCTGTGCTGGTCGTCT | ATGATGATAGCGACCGGCCCTCAGGGA, TCCCTGAGCGATTCTCTGGCTCCAACT CTGGGAACACGGCCACCCTGACCATCA GCAGGGTCGAAGCCGGGGATGAGGCC GATTATTACTGTCAGGTGTGGGATAGT GCTAGTGATCATTGGGTGTTCGGCGGA GGGACCAAGCTGACCGTCCTA GAGGTGCAGCTGTTGGAGTCTGGGGGÇ
AGGCTTGGTCCAGCCTGGGEGETCCCÇ 347 TGAGACTCTCCTGTGCAGCCTCTGGAT | BCMA-18 scFv (nt) TCACGTTTAGTAGCTATTGGATGAGCTG GCACCGCCAGGCTCCAGGGAAGGGGC
[57 | SeeuêneA The month CEGAGTGGGTEGGCCCACATAAACCAAG ACGGAAGTGAGAAGTACTATGTGGACT CTGTGAAGGGCCGATTCACCATCTCCA GAGACAACGCCGAGAGTTCACTGTATC TGCAAATGAACAGCCTGAGAGCCGAGG ACACGGCTGTGTATTACTGTGCGAGGT GGCTGGCGGTTACTAACTGGGGCCAG GGAACCCTGGTCACCGTCTCCTCAGGT GGAGGCGGTTCAGGCGGAGGTGGCTC TGGCGETGGCGGATCGCAGTCTGTGTT GACTCAGCCACCCTCAGCGTCTGGGAC ACPCGEGCAGAGGGTCACCATCTCTTG TTCTGGAAGCGGCTCCAACATCGGAAG TAATGATGTCTCCTGGTATCAGCAGATC CCAGGGACGGCCCCCAAACTCCTCATC TACTGGAATGATCAGCGGCCCTCAGGEG GTCCCTGACCGATTCTCTGGCTCCAAG TCTGGCACCTCAGCCTCCCTGGCCATC AGTGGGCTCCGGTCCGAGGATGAGGEC TGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTTGGGTGTTCGGCGG AGGGACCAAGCTGACCGTCCTA CAGGTCCAGCTGGTACAGTCTGGGGCT GAGGTGAAGAAGCCTGGGGCCTCAGT GAAGGTCTCCTGCAAGGCTTCTGGATA CACCTTCACCAGCTACTATATGCACTG GGTGCGACAGGCCCCTGGACAAGEGC TTGAGTGGATGGGATGGATCAACCCTA ACAGTGGTGGCACAAACTATGCACAGA AGTTTCAGGGCAGGGTCACCATGACCA GGGACACGTCCATCAGCACAGCCTACA
TGGAGCTGAGCAGGCTGAGATCTGACG 348 ACACGGCCGTGTATTACTGTGCGAGAG | BOMA-19 SFvY (nt) ATGGTGGGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCCTCAGGTGGA GGCGGTTCAGGCGGAGGTGGCTCTGEG CGGTGGCGGATCGCAGGCTGTGCTGA CTCAGCCTGCCTCCGTGTCTGGETCTC CTGGACAGTCGATCACCATCTCCTGCA CTGGAACCAGCAGTGACGTTGGTGATT ATAACTATGTCGCCTGGTATCAACAACA CCCAGGCAAMAGACCCCAAACTCATGAT
[57 | seeuênea the aeseão TTTTGAGGTCATTAATCGGCCCTCAGE GGTTTCTGATCGCTTCTCTGGCTCCAA GTCTGGCAACACGGCCTCCCTGGACAT CTCTGGGCTCCAGCCTGAGGACGAGG CTGATTATTACTGCATCTCATATTCACG AGGCAGCACTCCTTATGTCATCGGAAC TGGGACCAAGGTGACCGTCCTA GAGGTGCAGCTGTTGGAGTCTGGGGG AGGCTTGGTACAGCCTGGCAGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTTGATGATTATGCCATGCACTGG GTCCGGCAAGCTCCAGGGAAGGGCCT GGAGTGGGTCTCAGGTATTAGTTGGAA TAGTGGTAGCATAGGCTATGCGGACTC TGTGAAGGGCCGATTCACCATCTCCAG AGACAACGCCAAGAACTCCCTGTATCT GCAAATGAACAGTCTGAGAGCTGAGGA CACGGCCTTGTATTACTGTGCAAAGGG GGGCCTAGGAATAACCCCATACTACTT
TGACTACTGGGGCCAGGGAACCCTGGTI 349 - | CACCGTCTCCTCAGGTGGAGGCGGTTC | BCMA-20 scFv (nt) AGGCGGAGGTGGCTCTGGCGGTGGCG GATCGCAGCCTGTGCTGACTCAGCCAC CCTCAGCGTCTGGGACCCCCGGECAG AGGGTCACCATCTCTTGTTCGGGAGEC AAGACTGTAAACTGGTTCCGGCAGGTC CCAGGAACGGCCCCCCAACTCCTCATC TATAGTAATGATCAGCGGCCCTCAGGG GTCCCTGACCGATTCTCTGGCTCCAAG TCTGGCTCCTCAGCCTCCCTGGACATC AGTGGGCTCCAGTCTGAGGATGAGGCT TATTATTACTGTGGATCATGGGATGACA GCCTCAATGCTTGGGTGTTCGGCEGAG AGACCAAGCTGACCGTCCTA GAAGTGCAGCTGGTGGAGTCTGGEGE AGGCTTGGTCAAGCCTGGAGEGTCCCT GAAACTCTCCTGTGCAGCCTCTGGATT
CACCTTCAGTGACTACTACATGAGCTG 35º - | gaTCCGCCAGECTCCAGEGANGGEGEC | SºFY of BOMA-21 (nt) TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAGTACCATATACTACGCAGACTCT GTGAAGGGCCGATTCACCATCTCCAGG
Pee Tee epitope of human BCMA TFG
PCT AA 436 and ESA FG -44, -51 (aa) FS (aa) agctatgagctgacacagcctecaagegectetagcaca ccectggacagegagtgacaatgagctgtageggcaccag cagcaacatcggcagccacagegitgaactagtatcage agctgccectggcacageccectaaactgctgatctacacca acaaccagcggcctageggcgtgcceegatagatttteta gcagcaagagcggcacaagegecagectagcetatttet ggactgcagagcegaggacgaggccgactattattgtgcc | gcectgggacggcetetetgaacagecttatttttagcagagg caccaagctgacagtgctgggatctagaggtageggag 440 gatctggcggeggaggaageggaggeggeggatcetett | | BOMA-S52 (nt) scFv gaaatggctgaagigcagctagtgcagitctggegecga | (O / SSE) agtgaagaagcctggegagagcctgaagatcagetge aaaggcagcggctacagcttcaccagetactggategg ctgggtcegacagatgcctageaaaggcctigagtggat gggcatcatctacaceggegacacagegacaccagatac gcccetagcettteagggecacgtgaccatcagegecgaca agtctatcagcacegectacetacagtggtecagectaaa | ggcctetgacacegecatgtactactgegccagatactet ggacagcttcgacaattagggccagggcacactagtcac cgtgtccage ECT
ELI JR TE - = - IGSHSVNWYQQLPGTAPKLLIYTNNORPS | GVPDRFSGSKSGTSASLAISGLQOSEDEA
DYYCAAWDGSLNGLVFGGGTKLTVLGS 442 RGGGEGSCGSESEGESCGGGEGSLEMAEVOLVOQ | BCMA-52 scFv (aa) SGAEVKKPGESLKISCKGSGYSFTSYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSP SFQGHVTISADKSISTAYLQWSSLKASDT
AMYYCARYSGSFDNWGOQGTLVTVSS cagtctgccctgacacagectgccagegttagtactagte ceggacagtetategecateagetgtaceggcaccaget ctgacgttggctggtatcagcagcaccctggeaaggece ctaagctgatgatctacgaggacagcaagaggcecage ggcgtgatecaatagattcageggcagcaagageggcaa scFv BCMA-55 (nt) 460 caccgccagcectgacaattageggactgcaggeegag gacgaggccgattactactgcagcagcaacacceggte (0 / SSE) cagcacactggtttttygcggaggcaccaagetgacagt gctgggatctagaggtggeggaggatctggeggeggag gaagcggaggcggceggatctetigaaatggctgaagta cagctggtgcagtctggegecgagatgaagaaaccetag
Bread =. cttcategactactacgtgtactagatgcggcaggeccecet ggacagggactegaatctatgggctggatcaaccecaat agcggeggcaccaattacgcecagaaattceagggca gagitgaccatgaccagagacaccagcatcagcacege ctacatggaactgagceggctgagatcegacgacaceg ccatgtactactgcgecagateteagegegacggetacat) ggattattagggccagggaaccctagtcacegigtecag c - == - GWYQQHPGKAPKLMIYEDSKRPSGVSN RFSGSKSGNTASLTISGLQOAEDEADYYC
SSNTRSSTLVFGGGTKLTVLGSRGGGGS 478 GGGGSCGGGGSLEMAEVQLVOSGAEMK | BCMA-55 scFv (aa) KPGASLKLSCKASGYTFIDYYVYWMRQA PGQGLESMGWINPNSGGTNYAQKFOQGR VTMTRDTSISTAYMELSRLRSDDTAMYY CARSQRDGYMDYWGQGTLVTVSS
BCMA-49 WYOQKPGOAPVLVMS FR2 VL (aa) BCMA-51 WYQQKPGKAPKLLIF FR2 VL (aa) 483 GVPDRFSGSGSGTDFTLTISSLQAEDVAI FR3 VL of BCMA-24 (aa)
YHC 484 GVPSRFRGTGYGTEFSLTIDSLQOPEDFAT FR3 VL of BCMA-25 (aa) Wc 485 GVPDRFSGSGSGTDFTLKISRVEAEDVG FR3 VL of BCMA-28 (aa) WVWYCc 486 GVSNRFSGSGLGNAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
DYYC 187 GIPDRFSGSGSGTDFTLTISRLEHEDFAV FR3 VL of BCMA-30 (aa)
YYR 488 GVPDRFSGSNSGNTATLTVRGVEAGDEA | BC3-32 FR3 VL (aa)
DYYC GIPARFSGSGSGTDFTLTISSLEPEDFAV | pg3 vi of BCMA-34 (aa) Wc WTPARF: LLGGKAALTLSGAQPEDEA 490 SGSLLGGS SGAQ FR3 VL of BCMA-36 (aa)
DYYC 491 WTPARFSGSLLGGKAALTLSGAQPEDEA FR3 VL of BCMA-37 (aa)
EYYC 492 WTPARFSGSLLGGKAALTLSGAQPEDEA FR3 VL of BCMA-38 (aa)
DYFC GVPSRFSGSGSGTDFALTIRSLOPEDFAT] pn3 BCMA-39 (aa) Wc 494 GVPDRFSGTKSGTSASLAIRGLQOSDDDA FR3 VL of BCMA-41 (aa)
HYYC 495 GVPSRFSGSRSGTDYTLTISSLOPEDVAT VL FR3 BCMA-42 (aa) gaggtgcagctagigcagtctagagcagaggtgaaaaa toilet gceceggggagtetetgaagatctectataagggttctggat acagctttaccagctactggatcggctaggatacgccagat gecegggaaaggacctagagtggatggagatcatetatec VH chain BCMA-52 496 tagtgactctgataccagatacagccegtecttecaagge (nd) cacgtcaccatctcagctgacaagtcecatrageactgect acctgcagtggagcagcctgaaggecteggacacegoec | atgtattactgtgcgcgctactctagttctttogataactagg gtcaaggtactctggtgaccgtctecteage 197 GVPSRFSGSGSGTEFTLTISGVOSEDSA FR3 VL of BCMA-45 (aa)
TYHC GIPERFSGSKSGDTASLTISGVEAGDEAD | BCMA-47 FR3 VL (aa)
E 199 GIPERFSGSNSGNTATLTISRVEAGDEGD | 53) q BCMA-48 (aa) YYc 500 GIPERFSGSNSGNTATLTISRVEAGDEAA | 123 v, from BCMA-49 (aa) YYc 501 GVPSRFSGSCSGTDFTLTISSLQPEDVAV | 53) q BCMA-51 (aa) rc 504 BCMA-31 FGQGTKLDIK FR4 VL, BCMA-35 (aa) BCT-35 FGTGTKLDIK FR4 VL BCMA-42 (aa) BCMA- CDR-H1 FR4 VL 52 (aa) - Kabat numbering 508 FGQGTKVEIK FR4 VL of BCMA-24, -28, -51 (aa) BCMA-30 CDR-H1 (aa) BCMA-28 CDR-H2 (aa) GISWNSGSIX APM CDR numbering BCMA-33 -H2 (aa) BCMA-41 CDR-H2 YISSSGNTIY ADM Numbering 513 - | BCY-S IYPGDSDTRYSPSFQOG CDR-H2 (aa) - BCMA-28 Kabat CDR-H2 numbering (aa) SWNSG BCMA-33 Chothia CDR-H2 numbering (aa) SescesT BCMA-41 Chothia CDR-H2 numbering SSSGNT Chothia CDR-H3 numbering of BCMA-52 (aa) 517 YSGSFDN - Numbering of Kabat, Chothia and ADM EVOLVOSGGGLVKPGGSLRLSCAASGFT: 518 FSDYYMSWIRQAPGKGLEWVSGISWNS. BCMA-24 VA GSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCARDLGPPYGDDAFDIW GQGTMVTVSS
EVOLVOSGGGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA: 519 YGGTTEYAASVKGRFTISRDDSKSIAYLQ odea vi a) MA-25, - MNSLKTEDTAVYYCAAWSAPTDYWGQG | * 77 TLVTVSS
EVOLLESGGGLVQPGRSLRLSCVASGFT FDDYAMHWVRQAPGKGLEWVSGISWNS: 520 GSIGYADSVKGRFTISRDNAKNSLYLQMN the VH of BOMA-27 SLRAEDTALYYCAKGGLGITPYYFDYWG QGTLVTVSS
QVALVOSGGGLVOPGRSLRLSCAASGF TFDDYAMHWVRQAPGKGLEWVSGISWN | 521 SGSIXYADSVKGRFTISRDNAKNSLYLQM VA VA of BCMA-28 NSLRAEDTAVYYCARDLGPPYGDDAFDI GGQAGTMVTVSS
QVALVASGGGLVAPGRSLRLSCAASGF TFDDYAMHWVRQAPGKGLEWVSGISWN | 522 SGSIGYADSVKGRFTISRDNAKNSLYLQM PANA of BCMA-29, - NSLRAEDTAVYYCARDLGPPYGDDAFDI WGQGTMVTVSS
QVALVOSGGGLVOPGRSLRLSCAASGF TFGDYAMHWVRQAPGKGLEWVSGISWN | 523 SGSIGYADSVKGRFTISRDNAKNSLYLQM. BCMA-30 VA NSLRAEDTAVYYCARDLDPDDAFDIWGQ GTMVTVSS
QVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS | 524 TIYYADSVKGRFTISRDNAKNSLYLQMNS BOMA-S2 date, - LRAEDTAVYYCAKVDGPPSFDIWGQGTM VTVSS
QVALVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISGSGS: 525 TIYYADSVKGRFTISRDNAKNSLYLQMNS as VH of BOMA-3 LRAEDTAVYYCAREADSSADYWGQGTL VNVSS
TGQLVASGGGLVAPGRSLRLSCAASGFT 526 FDDYAMHWVRQAPGKGLEWVSGISWNS | BCMA-34 GSIGYADSVKGRFTISRDNAKNSLYLQMN VH chain | (aa) SLRAEDTAVYYCARDLGPDYDPDAFDIW
| | GoGTMVTVSS Po |
EVOQLVOASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS: 527 TIYYADSVKGRFTISRDNAKNSLYLQMNS. already BCMA-35 VA LRAEDTAVYYCARVDGDYDDYWGQGTL VTVSS
QVOLVOSGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS | 528 TIYYADSVKGRFTISRDNAKNSLYLQMNS aaa of BCMA-S6, LRAEDTAVYYCARVDGDYVDDYWGOGT LVTVSS
EVOLVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS | 529 TIYYADSVKGRFTISRDNAKSSLYLQMNS the VH of BOMA-37
LRAEDTAVYYCARVDGDYVDDYWGQGT LvTVSS QVALVOSGGGLVKPGGSLRLSCAASGFT) FSDYYMSWIRQAPGKGLEWVSYISSSGN | 530 - | TIVYADSVKGRFTISRDNAKNSLYLOMNS .. VH de BOMA-41 LRAEDTAVYYCAKVDGDYVDDYWGOGT LVTVSS
EVOLLESGGGLVQPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA: 531 YGGTTEYAASVKGRFTISRDDSKSIAYLQ the VH of BOMA-42 MNSLKTEDTAVYYCAAWSAPTDYWGQOG
BCMA-52 TLVTVSS CDR-H1 (aa) - Chothia Numbering
QVALLESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS: 533 TIYYADSVKGDSPSPGTTPKNSLYLQMN ia VH from BOMA-A7 SLRAEDTAVYYCAKVDGPPSFDIWROGT MVTVSS
DIVMTQSPDSLAVSLGERATINCKSSQSV 634 - | LYSSNNKNYLAWYQQKPGQPPKLLIVWG | BCMA-24 VL chain STRESGVPDRFSGSGSGTDFTLTISSLOA | (aa)
EDVAIYHCQQYISLPWTFGOGTKVEIK DIQMTQSPAFLSASVGDRVTVTCRASQG | 535 - | SNYLAWYQOKPGNAPRLLIYSASTLOSG | BCMA-25 VL chain VPSRFRGTGYGTEFSLTIDSLQPEDFATY | (aa) YCOOSYTSROTFGPGTRLDIK
Res a
SYVLTQPPSVSVAPGQTARITCGANNIGS 536 KSVHWYQAQKPGQAPMLVVYDDDDRPSG | BCMA-26 chain VL IPERFSGSNSGNTATLTISGVEAGDEADY | (aa) FCHLWDRSRDHYVFGTGTKLTVL
DIVMTQOSPLSLSVTPGEPASISCRSSOSL 537 LHSNGYNYLDWYLQKPGQSPQLLIYLGS | BCMA-28 VL chain NRASGVPDRFSGSGSGTDFTLKISRVEA | (aa) EDVGVYYCMQALQTPPWTFGQGTKVEIK |
QPVLTQPASVSGSPGOSITISCTGTSSDV 538 GSYNLVSWYQQHPGKAPKLMIYEVSKRP | BCMA-29 chain VL SGVSNRFSGSKSGNTASPTISGLQAEDE | (aa) ADYYCCSYAGSSTSRDVFGXGTKLTVL
EIVLTAOSPATLSVSPGERATLSCRASQPI 539 RSNLAWYQQKPGQAPKLLIYSASTRATGI | BCMA-30 VL chain PDRFSGSGSGTDFTLTISRLEHEDFAVYY | (aa) RRHYAPLTFGGGTKVEIK
DVVMTOSPDSLAVSLGERATISCKSSQOS 540 VLNSSNNKNYVAWYKQKPGQPPKLVISW | BCMA-31 VL chain ASTRESGVPDRFSGSGSGTDFTLTISSLOQ | (aa) AEDVAVYYCQQYYSTPYTFGQGTKLDIK
QTVVTAPPSVSVAPGQTARITCGGNNIG 541 SKGVHWYRQRPGQAPEVVIYDDSDRPS | BCMA-32 VL chain GVPDRFSGSNSGNTATLTVRGVEAGDEA | (aa)
DYYCQVWDSSSDHWVFGGGTKLTVL EIVMTOSPATLSLSPGDRATLSCRASOQS! 542 SNYLAWYQQKPGQAPRLLIYDASNRATG | BCMA-34 chain VL IPARFSGSGSGTDFTLTISSLEPEDFAVYY | (aa) CQAQARSNWPPYTFGQOGTKLDIK
NFMLTQPPSVSVAPGQTARITCGANNIGS 543 KSVHWYQQKPGQAPMLVVYDDDDRPSG | BCMA-35 VL chain IPERFSGSNSGNTATLTISGVEAGDEADY | (aa) FCHLWDRSRDHYVFGTGTKLDIK
QSVLTQEPSLTVSPGETVTLTCGSSTGP 544 VTSAHSPSWFQKKPGQAPTTLIYETTNR | BCMA-36 HSWTPARFSGSLLGGKAALTLSGAQPED VL chain | (aa) EADYYCLLSSGDARMVFGGGTKLTVL
QLVLTQEPSLTVSPGGTVTLTCGSSTGA 545 VTNGHSPYWFQQKPGQAPRTLIYDTTNR | BCMA-37 HSWTPARFSGSLLGGKAALTLSGAQPED VL chain | (aa) EAEYYCSLSHAGDRVFFGGGTKLTVL
[RPE ame fee VTNGHSPYWFQQKPGQAPRTLIYDTNNR | (aa) HSWTPARFSGSLLGGKAALTLSGAQPED
EADYFCLLSYSDARLAFGGGTKLTVL DIOXTAOSPSSLSASVGDRVTITCRASQGI | 547 RYELXWYQQKPGKAPKLLIYAASTLOSG | BCMA-39 VL chain VPSRFSGSGSGTDFALTIRSLQPEDFATY | (aa) YCLQHNSYPLTFGRGTKLEIK
QSALTQPASVSGSPGOSITISCTGSSSDV 548 SKYNLVSWYQQPPGKAPKLIIYDVNKRPS | BCMA-40 VL chain GVSNRFSGSKSGNTATLTISGLOGDDEA | (aa) DYYCCSYGGSRSYVFGTGTKLTVL
QPVLTQPPSVSGTPGQRVTIPCSGSSSNI 549 GGNSVDWFQEVPGTAPKLLIYANDRRPS | BCMA-41 VL chain GVPDRFSGTKSGTSASLAIRGLQSDDDA | (aa)
HYYCESWDDALNGHVFGGGTKLTVL DIQMTQSPSLVSASVGDRVTITCRASQG | 550 GNGLAWYQQKPGKAPKLLLFAASRLESG | BCMA-42 VL chain VPSRFSGSRSGTDYTLTISSLQPEDVATY | (aa)
BCMA-52 YCQQYVEDALTFGGGTKVDIK CDR-H2 (aa)
DVVMTOSPDSLAVSLGERATINCKSSQN 552 LLYSSNNKNYLAWYQQKPGQPPKLLIYW | BCMA-44 VL chain ASTRESGVPDRFSGSGSGTDFTLTISSLOQ | (aa) AEDVAVYYCQQYYSSPYTFGQGTKLEIK
AIRMTQOSPSSLSASVGDRVTITCRASQGI 553 GRSLAWYKQKPGGVPQLLIHDASSLRSG | BCMA-45 VL chain VPSRFSGSGSGTEFTLTISGVQSEDSATY | (aa) HCQAQQLNGYPWTFGQGTKVDIK
QAVLTQPPSVSVAPGKTATITCGGNNIGS 554 KSVHWYQARKPGQGPVVVIQYDTDRPSGI | BCMA-47 VL chain PERFSGSKSGDTASLTISGVEAGDEADY | (aa) YCQLWDSDSDDFAFGTGTKLTVL
QPVLTQPPSVSVAPGKTATITCGGNNIGS 555 KSVHWYQARKPGQGPVVVIQYDTDRPSGI | BCMA-48 VL chain PERFSGSNSGNTATLTISRVEAGDEGDY | (aa) YCQVWDSSSDHWVFGGGTKLTVL
LPVLTQPPSVSVAPGKTARITCGGDOIGR 556 KSVHWYQQKPGQAPVLVMSYDSDRPSG | BCMA-49 VL chain IPERFSGSNSGNTATLTISRVEAGDEAAY | (aa) YCQVWDSSTGQYVVFGGGTKLTVL
Eee Tae AIQLTOSPSTLSASVGDRVAITCRASQNI! 557 - | GDWLAWYQOKPGKAPKLLIFGASILESG | BCMA-51 VL chain VPSRFSGSGSGTDFTLTISSLOPEDVAVY | (aa) YCQKYDGAPPWTFGQGTKVEIK EVOLVOSGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSGISWNS
GSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCARDLGPPYGDDAFDIW scFv q q 558 GQOGTMVTVSSGGEESCEEESCGEGSD BOMA-24 (9a) fly
IVMTOSPDSLAVSLGERATINCKSSQSVL YSSNNKNYLAWYQQKPGQPPKLLIYWGS | TRESGVPDRFSGSGSGTDFTLTISSLOAE DVAIYHCQQYISLPWTFGAGTKVEIK EVOLVOSGGGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA
YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGQG seFv d 559 TLVTIVSSGGGESCGEGESCEGESDIAMT BOMA-25 (9a) fly QSPAFLSASVGDRVTVTCRASQGISNYL
AWYQQKPGNAPRLLIYSASTLOSGVPSR FRGTGYGTEFSLTIDSLQPEDFATYYCQA | SYTSROTFGPGTRLDIK EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAKVDGPPSFDIWGOGTM seFv d 560 VTVSSGGGESCECCESCEGESSYVLTAP | BOMA-26 (aa) fly PSVSVAPGOQTARITCGANNIGSKSVHWY QQKPGQAPMLVVYDDDDRPSGIPERFS GSNSGNTATLTISGVEAGDEADYFCHLW DRSRDHYVFGTGTKLTVL EVOQLLESGGGLVQPGRSLRLSCVASGFT FDDYAMHWVRQAPGKGLEWVSGISWNS
GSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTALYYCAKGGLGITPYYFDYWG scFv d 561 QGTLVTVSSGGGESCEGESCGEGESAP BOMA-27 (aa) flight VLTQPPSASGTPGQRVTISCSGGKTVNW FRQVPGTAPQLLIVYSNDORPSGVPDRFS GSKSGSSASLDISGLOSEDEAYYYCGSW
DDSLNAWVFGGETKLTVL TFDDYAMHWVRQAPGKGLEWVSGISWN | BCMA-28 (aa)
Fa ee SGSIXYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARDLGPPYGDDAFDI GGAGTMVTVSSGGEESCEGESCAGES DIVMTOSPLSLSVTPGEPASISCRSSQSL LHSNGYNYLDWYLQKPGOSPOQLLIYLGS
NRASGVPDRFSGSGSGTDFTLKISRVEA EDVGVYYCMQALQTPPWTFGQGTKVEIK | QVALVASGGGLVAPGRSLRLSCAASGF TFDDYAMHWVRQAPGKGLEWVSGISWN
SGSIGYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARDLGPPYGDDAFDI e F 563 WGQGTMVTVSSGGGESCEGESCEGGE AND CMASS (da) vde SQPVLTQPASVSGSPGOSITISCTGTSSD VGSYNLVSWYQQHPGKAPKLMIYEVSKR PSGVSNRFSGSKSGNTASPTISGLOAED EADYYCCSYAGSSTSRDVFGXGTKLTVL QVALVASGGGLVAPGRSLRLSCAASGF TFGDYAMHWVRQAPGKGLEWVSGISWN
SGSIGYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARDLDPDDAFDIWGQ | dwarf of scFv 564 - | GTMVTVSSGGGESCECESGEGESEIVL | SA 30 (aa) TQSPATLSVSPGERATLSCRASQPIRSNL AWYQQKPGQAPKLLIYSASTRATGIPDRF SGSGSGTDFTLTISRLEHEDFAVYYRRHY APLTFGGGTKVEIK EVOLVOSGGGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA
YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGQG seFv d 565 TLVTVSSGGGESCEGESCEGESDVVM BOMA-S1 (aa) fly TQSPDSLAVSLGERATISCKSSQSVLNSS NNKNYVAWYKQKPGQPPKLVISWASTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQOQYYSTPYTFGQGTKLDIK QVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS 566 LRAEDTAVYYCAKVDGPPSFDIWGQGTM | VTV scFv sequenceSSGGEGESCECESCEGESATVVTA | BCMA-32 (aa) PPSVSVAPGOTARITCGGNNIGSKGVHW YRORPGOQAPEVVIYDDSDRPSGVPDRFS GSNSGNTATLTVRGVEAGDEADYYCQV
[67 | seeuênea the cleanliness |] WpsssDHWVFGGGTRETVE | QVALVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISGSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAREADSSADYWGQGTL and F 567 VNVSSGGGESCEGESGEGESAPVLTA Seas (da) vde PPSVSVAPGKTAMITCGGNNIGFKGVQW YQQAKTGQAPVLVVYDDSDRPSGIPERFS GSNSGNTATLTISRVEAGDEADYYCQVW DSASDHWVFGGGTKLTVL TGQLVASGGGLVAPGRSLRLSCAASGFT FDDYAMHWVRQAPGKGLEWVSGISWNS
GSIGYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCARDLGPDYDPDAFDIW scFv q 568 GQEGTMVTVSSGEGGESCEGESCEGESE BOMA-S4 (aa) fly IVMTQOSPATLSLSPGDRATLSCRASOSIS NYLAWYQQKPGQAPRLLIYDASNRATGI! PARFSGSGSGTDFTLTISSLEPEDFAVYY CQAORSNWPPYTFGQGTKLDIK EVOLVOSGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARVDGDYDDYWGQGTL seFv d 569 VTVSSGGEGESCECCESCGECGESNFMLTA BOMA-35 (aa) fly PPSVSVAPGOTARITCGANNIGSKSVHW YQQKPGQAPMLVVYDDDDRPSGIPERFS GSNSGNTATLTISGVEAGDEADYFCHLW DRSRDHYVFGTGTKLDIK QVALVASGGGLVKPGGSLRLSCAASGFT) FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARVDGDYVDDYWGQGT ency of scFv d 570 LVTVSSGGGESCEGESCEGESASVLTA | BOMA-S6 (aa) flight EPSLTVSPGETVTLTCGSSTGPVTSAHSP | SWFQKKPGQAPTTLIVETTNRHSWTPAR FSGSLLGGKAALTLSGAQPEDEADYYCL LSSGDARMVFGGGTKLTVL
EVOLVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS seFv d 571 TIYYADSVKGRFTISRDNAKSSLYLQMNS BOMA-S7 (aa) fly LRAEDTAVYYCARVDGDYVDDYWGQGT LVTVSSGGGESEEGGESCEGESALVLTA
[57 | seeuêne the eesgiving EPSLTVSPGGTVTLTCGSSTGAVTNGHS PYWFQQKPGQAPRTLIYDTTNRHSWTPA RFSGSLLGGKAALTLSGAQPEDEAEYYC SLSHAGDRVFFGGGTKLTVL QVALVASGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCARVDGDYVDDYWGAGST | 5-1 51 5ia soFv 572 | IVIVSSGGGESCEGESEEGESANAVLTA | Ze, 38 (aa) EPSLTVSPGGTVTLTCGSSTGAVTNGHS PYWFQQKPGQAPRTLIYDTNNRHSWTP ARFSGSLLGGKAALTLSGAQPEDEADYF CLLSYSDARLAFGGGTKLTVL QVALVASGGGLVAPGRSLRLSCAASGF TFDDYAMHWVRQAPGKGLEWVSGISWN
SGSIGYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARDLGPPYGDDAFDI | ..0 scFv age of 573 | WGAGTMVTVSSGEEESCEGESEEEE | AA, 39 in) SDIQXTASPSSLSASVGDRVTITCRASQG IRYELXWYQQKPGKAPKLLIYAASTLOSG VPSRFSGSGSGTDFALTIRSLQPEDFATY YCLOHNSYPLTFGRGTKLEIK EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAKVDGDYTEDYWGQOGT seFv d 574 LVTVSSGGGESCEGESCEGESASALTA | BOMA-4O (aa) fly PASVSGSPGOSITISCTGSSSDVSKYNLV SWYQQPPGKAPKLIIYDVNKRPSGVSNR FSGSKSGNTATLTISGLOGDDEADYYCC SYGGSRSYVFGTGTKLTVL QVALVOSGGGLVKPGGSLRLSCAASGFT) FSDYYMSWIRQAPGKGLEWVSYISSSGN
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAKVDGDYVDDYWGQOGT seFv d 575 LVTVSSGGGESCEGESCECGESAPVLTA BOMA-A1 (aa) flight PPSVSGTPGQRVTIPCSGSSSNIGGNSV DWFQEVPGTAPKLLIVANDRRPSGVPDR FSGTKSGTSASLAIRGLOSDDDAHYYCE
SWDDALNGHVFGGGTKLTVL FGDYAMSWFRQAPGKGLEWVGFIRSKA | BCMA-42 (aa)
Fe eee YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGOG TLVTIVSSGGGGSCGGESGEGGSDIAMT QSPSLVSASVGDRVTITCRASQGIGNGLA WYOQKPGKAPKLLLFAASRLESGVPSRF SGSRSGTDYTLTISSLOPEDVATYYCQQ
YVEDALTFGGGTKVDIK CDR-H1 from BCMA-52 (aa) EVOLVOSGGGLVQPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA
YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGOG ency of scFv q 578 TLVTVSSGGGESCEGESCGEGESDVVM GOOD Aa and scrv of TQSPDSLAVSLGERATINCKSSQNLLYSS (44) NNKNYLAWYQQKPGQPPKLLIVWASTRE SGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQOYYSSPYTFGOGTKLEIK QVALVASGGEGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA
YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGQG ae 579 TLVTIVSSGGGESCEGESGEGGSAIRMT GOOD AS scFv from QSPSSLSASVGDRVTITCRASQGIGRSLA 45 aa) WYKOKPGGVPOLLIHDASSLRSGVPSRF SGSGSGTEFTLTISGVAOSEDSATYHCQQ LNGYPWTFGQGTKVDIK QVQLLESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGDSPSPGTTPKNSLYLQMN SLRAEDTAVYYCAKVDGPPSFDIWROGT ae 580 MVTVSSGGGESGEGESGECEGSAAVLT SAVADAS QPPSVSVAPGKTATITCGGNNIGSKSVH WYQORKPGQGPVVVIQYDTDRPSGIPERF SGSKSGDTASLTISGVEAGDEADYYCQL WDSDSDDFAFGTGTKLTVL EVOLVESGGGLVKPGGSLRLSCAASGFT
FSDYYMSWIRQAPGKGLEWVSYISSSGS 581 TIYYADSVKGRFTISRDNAKNSLYLOMNS | BCMA-48 LRAEDTAVYYCAKVDGPPSFDIWGQGTM sequence | scFv from (aa) VTVSSGGGESCEGESCGEGSAPVLTA PPSVSVAPGKTATITCGGNNIGSKSVHW
[57 | seeuênea the pesenão YORKPGQGPVVVIAYDTDRPSGIPERFS GSNSGNTATLTISRVEAGDEGDYYCQVW DSSSDHWVFGGGTKLTVL QVQLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS
TIYYADSVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCAKVDGPPSFDIWGASTM | 5-7 21 5ia scFv 582 - | VIVSSGGGESCEGESCEGESLPYLTAP | Rol, 9 (aa) PSVSVAPGKTARITCEGGDQIGRKSVHWY QQKPGQAPVLVMSYDSDRPSGIPERFSG SNSGNTATLTISRVEAGDEAAYYCQVWD SSTGQYVVFGGGTKLTVL EVOLVOSGGGLVAPGRSLRLSCTASGFT FGDYAMSWFRQAPGKGLEWVGFIRSKA
YGGTTEYAASVKGRFTISRDDSKSIAYLQ MNSLKTEDTAVYYCAAWSAPTDYWGOG seFv d 583 TLVTIVSSGGGESCGEGESCGEGGESAIALT BOMA-S1 (aa) flight QSPSTLSASVGDRVAITCRASQNIGDWL AWYQQKPGKAPKLLIFGASILESGVPSRF SGSGSGTDFTLTISSLAPEDVAVYYCQKY DGAPPWTFGQGTKVEIK CAGGTGCAGCTGGTGCAATCTGGGEG AGGCTTGGTCAAGCCTGGAGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGATT CACCTTCAGTGACTACTACATGAGCTG GATCCGCCAGGCTCCAGGGAAGGGGC TGGAGTGGGTTTCATACATTAGTAGTAG TGGTAATACCATATACTACGCAGACTCT GTAAAGGGCCGATTCACCATCTCCAGG
GACAACGCCAAAAACTCACTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGAC 1 584 ACGGCCGTGTATTACTGTGCGAAAGTG BOMAAT and scFv of GACGGTGACTACGTCGATGACTACTGG 41 (nt) GGCCAGGGAACCCTGGTCACCGTCTC CTCAGGTGGAGGCGGTTCAGGCGGAG GTGGCTCTGGCGEGTEGCGGATCGCAG CCTGTGCTGACTCAGCCACCCTCAGTG TCTGGGACCCCCGGGCAGAGGGTCAC CATCCCTTGTTCTGGAAGCAGCTCCAA CATCGGAGGTAACTCTGTAGACTGGTT CCAGGAGGTCCCAGGGACGGCCCCCA
AACTCCTCATCTACGCTAATGATCGGC month and GGCCCTCGGGTETCCCTGACCOGCTTCT CTGGCACCAAGTCGGGCACCTCAGCCT CCCTGGCCATCAGGGGGCTCCAGTCOT GACGATGACGCTCATTATTACTGTGAAT CCTGGGACGATGCCCTGAACGGTCAC GTGTTCGGCGGAGGGACCAAGCTGAC CGTCCTA QIQLVASGPELKKPGETVKISCKASGYTF TDYSINWVKRAPGKGLKWMGWINTETRE PAYAYDFRGRFAFSLETSASTAYLQINNL
KYEDTATYFCALDYSYAMDYWGQGTSV 585 - | TvsseccescecesceccesnivrTtasP | SºFY VH-VL from BOEMA-C1 PSLAMSLGKRATISCRASESVTILGSHLIH (aa) WYQQKPGQPPTLLIQLASNVATGVPARF SGSGSRTDFTLTIDPVEEDDVAVYYCLOS RTIPRTFGGGTKLEIK DVVMTOSHRFMSTSVGDRVSITCRASQD
VNTAVSWYQQKPGQSPKLLIFSASYRYT GVPDRFTGSGSGADFTLTISSVQAEDLAV |
YYCQQHYSTPWTFGGGTKLDIKGGGGS 586 | GeGescecesalaLvascPDLKKPGET | BEMA-C2 SSFY VL-VH VKLSCKASGYTFTNFGMNWVKQAPGKG | (9th) FKWMAWINTYTGESYFADDFKGRFAFSV ETSATTAYLQINNLKTEDTATYFCARGEIY
CDR-H2 YGYDGGFAYWGQGTLVTVSA BCMA-52 (aa) gaagtgcagctagtgcagtctagegecgaagtgaagaa gcctggegagagcctgaagatcagctgcaaaggeage ggctacagcttcaccagctactggateggctaggatecga cagatgcctggcaaaggccttigagtggataggcateatct | 588 accceeggegacagegacaccagatacagccctagcettt | BCMA-52 VM chain cagggccacgtgaccatcagegeegacaagtetateag | (nt) (O / SSE) cacegectacetacagtggtecagectagaaggcctetga caccgccatgtactactgcgccagatactctggcagcette gacaattggggccagggcacactggtcacegtgtecag c CDR-L1 BCMA-S2 (aa) 589 SGT
- Kabat, Chothia and ROM CDR-L3 BCMA-52 (aa) 591 AAWDGSLNGLV - Kabat, Chothia and ROM tcctatgagctgactcagecacecteagegtetgggaceo cegggcagagggtcaccatgtettattctagaaccagete caacatcggaagtcactctgtaaactggtaccagcaget cccaggaacggecccecaaactecteatetatactaataat VL chain BCMA-52 592 cagcggcccteaggggtecetgacegattetetggeteca (nd agtctggcaccteagectecetagecatcagtggecteca gtctgaggatgaggctgattattactgtacagcataggata gcagcctgaatggtctggtaticageggagggaccaagce | tgacegtectaggt 593 DYYVY CDR -H1 BCMA-55 (aa) - Kabat numbering 594 WINPNSGGTNYAQKFOG CDMA-SS of BCMA-SS (aa) - Kabat numbering CDR-H3 of BCMA-55 (aa) 595 SQRDGYMDY - Kabat numbering, Chothia and ADM CDR -H1 of BCMA-55 (aa) SYTFIDY - Numbering of Chothia CDR-H2 of BCMA-55 (aa) NPNSGG - Numbering of Chothia CDR-H1 of BCMA-55 (aa) SYTFIDYYVY - Numbering of APM CDR-H2 of BCMA- 55 (aa) WINPNSGGTN - APM Numbering agctatgagctgacacagcctecaagegectetageaca ccectggacagegagtgacaatgagctgtageggcaccag cagcaacatcggcagccacagegtgaac tagtateage agctgccectggcacagcccectaaactgctgatctacacca VL chain acaaccagcggcctageggcgtgccegatagattttcta BCMA-52 (nt) (O / SSE) gcagcaagageggcacaagegecagectagetatttet ggactgcagagcgaggacgaggccgactattattgtgcc | gcectgggacggctetetgaacgagcecttatttttagecggagg caccaagctgacagtgctggga BCMA-55 CDR-L1 (aa) 601 TGTSSDVG - Kabat, Chothia and ADM numbering
Peas Tee O e es Ee 602 EDSKRPS - Kabait, Chothia and ADM numbering and eee Eça 603 SSNTRSSTLV - Kabat, Chothia and ADM numbering [60 TARSSSEON | coORHSdeBCMASA (the
EVOLVASGAEVKKPGESLKISCKGSGYS DTRYSPSFOGHVTISADKSISTAYLQWSs | VH of BOMA-S2 LKASDTAMYYCARYSGSFDNWwGaGTLV | (9th)
TVSS the EE and 610 IGSHSVNWYQQLPGTAPKLLIYTNNORPS | BCMA-52 VL chain GVPDRFSGSKSGTSASLAISGLQOSEDEA | (aa) DYYCAAWDGSLNGLVFGGGTKLTVLG
EVOLVOAOSGAEMKKPGASLKLSCKASGYT 617 | GeTNYAGKFOGRVTITRDTSISTAYMEL | 2909 BCMA-S6 SRLRSRSDDTAMYYCARSORDGYMDYWwG | (9th)
QGTLVTVSS EEE eee 618 GWYQQHPGKAPKLMIYEDSKRPSGVSN | BCMA-55 chain VL RFSGSKSGNTASLTISGLQAEDEADYYC | (aa)
SSNTRSSTLVFGGGTKLTVLG gatctccggagcatacgga human cover (nt)
The ECT [hmana (j = | gaatctaagtacggacegeccetgacectecetgecetaete ctccetgtagetagaccaagegtattectatttecacctaago | ctaaagataccctgatgatttccegcacacetgaagtgac gcagttcaactagtacgtggacggcgtagaagitccacaa ttgcgtagtcgtagacgatgagecaggaggatccagaagt | tgctaagactaaacccegagaggaacagtttcagtcaac ttaccgggtegtgagegtgctgacegtectacatcaggatt DEAD DR AI Dar aM Nro area acs | g4 Spacer / 962
621 joint- IgG2 / I9G4 gcaaaagggcagcctegagaaccacaggtatatacccet
Cr2- I9gG4 Cr3 (nt)
gccecetagecaggaggaaatgaccaagaaccaggta teccectgacatgatetggtcaaaggactictatccaagigacat cgcegitggagtgggaatcaaatgggcagecegagaac aattacaagaccacaccacgggtggtggtggtggtggtggtggtgt agccctgcacaatcattatacacagaagtcactgagcect gitccctgggcaaa gagtctaaatacggacegcecttatectecttgtecegetect ccetgttgceggaccttecgtattectatitectecaaagecta aggacaccctgatgatcagcaggacccecctgaagtgace tacgtagtagtggatgtateccaagaggatccegaggta cagttcaactggtatgtagacggcgatagaagtgcacaac gccaagaccaagcctagagaggaacagttccagageca ccetacagagtggtatcecgtgctgacagtgctgcaccagg attggctgaacggcaaagagtacaagtgcaaggtatcce | IGG4 / I9G2 spacer
622 aacaagggcctacetagcagcategagaaaaccatete | hinge- IgG2 / I19G4 Ch2- caaggccaagggccagecaagagagcecccaggtttac | I9G4 Ch3 from SSE acactgcctccaagccaagaggaaatgaccaagaatc | Optimized (nt) aggtgtccctgacatgcctagteaagggcettetacceeetec gatatcgcegtggaataggagagcaatggccagectga gaacaactacaagaccacacctcctgtactggacageg acggcagtttcttectatatagtagactcaccgtagataaat caagatggcaagagggcaacgtgattcagetacagegta atgcacgaggccctgcacaaccactacacccagaaaa gcctgagcctgtetetaggcaag
623 atgttttaggtactagtegtggtegaagagatactagectat CD28 tacagcctactagtgacagtegctttcatcatecttetaggta (nd)
domain of
ECT fa Õj aage: agaaagaaactgctgtatattticaaaca: An Cootttatgagacclgtacagactacecaggaggaagae Sequencá de; 625 ggatacagctgtaggtiteccgaggaagaggaaggagg cossinalização derived from intracellular 4-1BB (nt) 626 ctgtgagcta KRGRKKLLYIFKAQPFMRPVQTTQEEDGC Sonsinatzação derived from intracellular SCRFPEEEEGGCEL 4-1BB (aa) agagtcaagttttecaggtecgeegacgetecagectace agcaggggcagaaccagctgtacaacgagctgaacct gggcagaagggaagagtacgacgtcctgagataagegg agaggcegggaccecetgagataggeggcaagectegge | signaling domain 627 ggaagaaccccecaggaaggacctatataacgaactgca | intracellular derived from gaaagacaagatggcegagacctacagegagatcgge! CD3-zeta (nt) atgaagggegageggaggeggggcaagggecacga cggcctgtateagggectatecacegecaccaaggatac ctacgacgccectgcacatgcaggeectgececcaaggg
RVKFSRSADAPAY QQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNP | signaling domain 628 QEGLYNELQKDKMAEAYSEIGMKGERRR | derived intracellular GKGHDGLYQGLSTATKDTYDALHMOQALP | CD3-zeta (aa)
PR [5 | stmgccsagttaenn Ugo emma 629 agcaatggaaccattatccatgtgaaagggaaacacctt CD28 (nt) tatecaagteccectattteceggaccttetaagece SPLFPGPSKP CD28 (aa) atgcttctcctagtgacaagcecttetactetatagagttacca cacccagcattectectgateccacgcaaagitgtataacg gaataggtattggtgaatttaaagactcactctccataaat gctacgaatattaaacacttcaaaaactgcacctccatca gtggegatctecacatcctaceggtageatttaggggtaa P8A EIS Macaw Orr rraaao SO] CGFR truncated sequence (tEGFR) (nt) attcaggcttggectgaaaacaggacggacctecatgee tttgagaacctagaaatcatacgcggcaggaccaagea acatggtcagttttctcttgcagtegtcagcectgaacataac atccttgggattacgcteccteaaggagataagtgataga gatgtgataatttcaggaaacaaaaatttgtactatgcaaa | tacaataaactggaaaaaactgtttaggacctceeggtca
[49 weighed seven gaaaaccaaaattataagcaacagaggtgaaaacage tgcaaggccacaggccaggtetaccatgccttgtactece cegagggcetactagggeceggageccagggactgacat ctcttgceggaatgteageegaggcagggaatgcgtaga caagtgcaaccttctagagggtgagecaaggagagtttat ggagaactctgagtgcatacagtgccacccagagtgccet gcectceaggecatgaacatcacctgcacaggacgggga ccagacaactgtatccagtgtgcecactacattgacggcc | ceccactgegtcraagacctgcceggcaggagtcatagga gaaaacaacaccctggtctagaagtacgcagacgecg gccatgtgtaccacctgatgccatccaaactagcacctacgg atgcactgggccaggtettgaagactgtecaacgaatgg gcectaagatccegtecategecactaggatagtagggge cetectettgctactagtagtagecctagggateggcctett catgtga atgctgctectegigacaagectgactectatatagaactecct catccagcttttetgcteattecteggaaagtatacaacgg catcggcatcggagagttcaaggacagcctgagcatca atgccaccaacatcaagcactticaagaattgcaccage atcagcggegacctgacacattctacctatagecttitagag gegacagcttcacccacacaccetecactggatececaa gagctggatatcctgaaaaccgtgaaagagattaccgg attcctectgatccaagectggecagagaacagaaceg atctgcacgccticgagaacctegagatcateagaggec ggaccaaacagcacggccagtttagcctagctatagtat ctctgaacatcaccagtctgggcctgagaagectagaaag aaatctecgacggegacgtgatcatetecggaaacaag aacctgtgctacgccaacaccatcaactggaagaaget | EGFR 633 sequence gtteggcacctecggecagaaaacaaagatcatctetaa | truncated (tEGFR) (nt) ceggggegagaacagetgcaaggecaceggacaagtt | (O / SSE) tgtcacgccctgtgtagecctgaaggctatiggggaceoeg aacctagagactgtgtatcctaceggaatgtatecegggg cagagaatgtgtggataagtgcaacctgctiggaaggcg agcceceegegagtttatagaaaacagegagtgacatecagt gtcaccceegagtgtetagceccaggccatgaacattacatg caccggcagaggcccegacaactgtaticagtgegece actacatcgacggcccteactgcgtgaaaacatgtccag ctggcgtgatgggagagaacaacaccctegtgtagaag tatgccgacgceggacatgtgtgccacctgtgteacceceta attgcacctacggctgtaccggacctggectagaaggat gcecetacaaacggccctaagatccccageattigecace ggaatggttagagccctgctacttctattagtagtiggcecte
ECT | - | ggaateggeetaticatatiga O Çj |
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGI GEFKDSLSINATNIKHFKNCTSISGDLHILP | VAFRGDSFTHTPPLDPQELDILKTVKEITG FLLIQAWPENRTDLHAFENLEIIRGRTKQH
GQFSLAVVSLNITSLGLRSLKEISDGDVIIS GNKNLCYANTINWKKLFGTSGQKTKIISN EGFR 634 sequence RGENSCKATGQVCHALCSPEGCWGPEP RDCVSCRNVSRGRECVDKCNLLEGEPR | runcado (tEGFR) (aa) EFVENSECIQCHPECLPQAMNITCTGRG PDNCIQCAHYIDGPHCVKTCPAGVMGEN NTLVWKYADAGHVCHLCHPNCTYGCTG PGLEGCPTNGPKIPSIATGMVGALLLLLV
VALGIGLFM ggatctgegategetecggtaceegtcagtaggcagage gcacatcegcccacagtececgagaagttaggagggagg gateggcaattgaaceggtgcctagagaaggtagegeg gggtaaactgggaaagigatgtcgtgtactggctecgect ttttccegagggtaggggagaaccgtatataagtgcagta gtcgcegtgaacgttetttttegcaacgggtttacegecag aacacagctgaagcttegaggggctegceatetetecttea | EF1alfa promoter with 635 cgcgecegeegecetacetgaggecgccatecacgaeeg | enhancer of HTLV-1 gttgagtcgcegttetacegectecegectatagtacetecta aactgcgtecgcecgtetaggtaagtttaaageteaggteg agaccgggcctttgtecggegceteccttggagectaceta gactcagcegactetecacgcetttacetgacectgacttget caactctacgtctttgtttegttttctatictacgccegttacaga tccaagcetgtgaceggegectac aatcaacctctggattacaaaatttgtgaaagattgactgg tattcttaactatgattgctecttttacgctatatagatacgctgc tttaatgcctttgtatcatgctattgcttcccgtatggctttcattt tctectecttgtataaatcctggttgctatetetitataaggagt tgtagccegttgteaggcaacatagegtagtatacactata tttgctgacgcaacccccactagttggggcattgccacca | 636 Hepatitis Virus cetgteagetectttecgggactttegetttececetecetatt | Woodchuck (WHP) gecacggeggaacteategecgectaccettacecgetae | Regulatory Element Post-tagacaggggcteggctattggacactgacaattcegtag! transcriptional (WPRE) tattgteggggaaatcategtectttectiggctactegecta tagttgccacctagattctacgegggacgtecttctactacgt cecetteggeceteaatecageggaccttecttecegegge ctgctgceggetetgeggectegecgettggegegggegggegggegg
ECT BCMA-52-scFV-mFc 637 QNEYF BCMA binding epitope 1 BCMA-52-scFV-mFc 638 CIPCQL BCMA binding epitope 2 BCMA-52-scFV-mFc 639 CORYC BCMA epitope 2 BCMA-55-scFV- mFc 640 MLMAG binding epitope 1 BCMA-55-scFV-mFc 641 binding BCM-55F scmvgmggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggf tacaccttcatcgactactatgtatactagatgcgacagge cccetggacaagggcettigagtccataggatggatcaaccec 643 taacagtggtggcacaaactatgcacagaagtttcaggg | BCMA-55 VM chain cagggtcaccatgaccagggacacgtecatcageaca | (Nt) gcctacatggagctgagcaggctgagatctgacgacac cgccatgtattactgtgcgegeteccagegtgacgagttac atggattactggggtcaaggtactctggtgaccgtetecte the gaagtgcagctggtgcagtctagegcegagatgaagaa acctggegcectetetagaagetgagetgcaaggecageg gctacaccttcategactactacgtgtactagatgcggea ggcccectggacagggactegaatetatgggctggatcaa ccecaatageggeggcaccaattacgcecagaaattce | BCMA-55 VM chain 644 agggcagagtgaccatgaccagagacaccagcatcag | (Nt) (O / SSE) cacecgcctacatggaactgagceggctgagatcegacg acacecgccatgtactactgcgccagatcteagegegaca gctacatggattattggggccagggaaccctagtcacegt gtccage caatctgccectgacteagectgccetecgtgtetgcgtetect VL chain BCMA-55 645 ggacagtcgatcgecatctectacactagaaccagceagt (nd gacgttggttggtatcaacagcacccaggcaaagecec
[49 caaactcatgatttatgaggacagtaageggceceteagg seen weighed ggatttctaategcttetetagetecaagtetageaacacgg cetecetgaccatetetaggctecaggctgaggacgagg ctgattattactgcagctcaaatacaagaagcagcacttto gtgtteggcggagggaccaagetgacegtecta cagtctgccctgacacagectgecagegttagtactagte ceggacagtetategecatcagetgtaccggcaccaget ctgacgttggctggtatcagcagcaccctggeaaggece ctaagctgatgatctacgaggacagcaagaggcecage VL chain BCMA-55
646 ggcgtgtecaatagatticageggcagcaagageggcaa
(nt) (O / SSE)
cacegecagcectgacaattageggactgcaggeegag gacgaggcegattactactgcagcagcaacacceggte cagcacactggtttttggecggaggcaccaagetgacagt getg tcctatgagctgactcagecacecteagegtetagggaceo cegggcagagggtcaccatgtettgttctagaaccagete caacatcggaagtcactctgtaaactggtaccagcaget cccaggaacggecccecaaactecteatetatactaataat cagcggcccteaggggtecetgacegattetetggeteca agtctggcaceteagectecetagecatcagtggecteca gtctgaggatgaggctgattattactgtacagcatgggata gcagcctgaatggtctagtattecggeggagggaccaagro | tgaccgtecctaggttctagaggtagtagtagtageggegg
647 cggcgactetagtagtagtagatccctegagatggecga | BCMA-52 scFv gatgcagctagtgcagtctaggagcagaggtgaaaaage ceggggagtetetgaagatectectgtaagggttctagatac: agctttaccagctactggateggctaggtacgccagatge cegggaaaggcctggggggggggggggggggggg cgtcaccatcteagetgacaagtecateageactgectac ctgcagtggagcagcctgaaggcecteggacacegecat gtattactgtgcgcegetactetagttetttoegataactgggagt caaggtactctggtgacegtetectea caatctgccectgacteagectacecetecgtatetagegtetect ggacagtegategecatetectgcactagaaccageagt gacgttggttagtatecaacagcacccaggeaaageceo caaactcatgatttatgaggacagtaagcggcccteagg
648 ggtttctaategettetetaggetecaagtetagcaacacgg | BCMA-55 scFvy cetecetgaccatetetaggetecaggectaaggacgagg ctgattattactgcagctcaaatacaagaagcagcacttta gtgtteggeggagggaccaagetgaccgtectagattcta gaggtagtggtggtageggagagggact
[49 gtggatccctegagatggcegaagtgcagetagtacagt semen weighed ctggggctgagatgaagaagectggggccteactgaag ctctectgcaaggcttctagatacaccttcategactactat gtatactggatgcgacaggccecetagacaagggcttigag tccatgggatggatcaaccctaacagtagtagcacaaac tatgcacagaagttteagggcagggtcaccatgaccagg gacacgtccatcagcacagcectacatagagetgageag gctgagatctgacgacaccgccatgtattactgtacgege teccagegtgacggttacatggattactagggtcaaggta ctctagtgacegtetectea ESKYGPPCPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQF
NWYVDGVEVHNAKTKPREEQFQSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPS | IgG4 / I9G2 Spacer 649 SIEKTISKAKGQPREPQVYTLPPSQEEMT | joint- I9G2 / I19G4 KNQVSLTCLVKGFYPSDIAVEWESNGOQP | Ch2- IgG4 CH3 (aa) ENNYKTTPPVLDSDGSFFLYSRLTVDKSR | WQEGNVFSCSVMHEALHNHYTQKSLSL
SLGK ggatctgegategetecggtgccegtragtaggcagage gcacatcegceccacagteceegagaagttaggggIgagg gateggcaattgaaceggtgcctagagaaggtggegeg gggtaaactgggaaagigatgtegtgtactggetecgect ttttccegagggtaggggagaaccgtatataagigcagta gtcgcegtgaacgttetttttegcaacgggtttacegecag 650 aacacagctgaagcttegaggggctegceatetetecttea | promoter EF1 alfa cgegecegecgecetacetagaggecgecatceacgeca | modified gttgagtcgegttetacegectecegectatagtgectecta aactgcgtecgecgtetagataagtttaaageteaggteg agaccgggcctttgtecggegetecetiggagectaceta gactcagceggctetecacgcetttacetgacectgactiget caactctacgtctttgtttogttttetatictacgcegttacaga tecaagcetatgaceggegectacggctagegec tttatttagtctecagaaaaaggggggaatgaaagacce cacctgtaggtttagcaagctaggatcaaggttaggaac agagagacagcagaatatgggccaaacaggatatctgt ggataagcagttcctgccceggctragggecaagaacag 651 ttggaacagcagaatatgggccaaacaggatatctgtag | MND promoter taagcagttcctgccceggctragggecaagaacagat gatecccagatagcggtecegeceteagreagtttetagaga | accatcagatgtttccagggtgcceccaaggacctgaaat gaccctgtaccttatttgaactaaccaatcagttegettete gettctgttegegegettetacteccegageteaataaaag agccca agagtgaagttcagcagatcegeegacgctecagectat cagcagggccaaaaccagcetgtacaacgagctgaace tgagggagaagagaagagtacgacgtgctggataageg acggaagastecicaagagagecigtataagageie | 2minio 652 9ggaas cattle gagggce'g'aaa intracellular signaling derivative agaaagacaagatggcegaggcctacagegagateg CD3-zeta (nt) gaatgaagggegagegcagaagaggcaagggacac gatggactgtaccagggcctgagcacegecacraagg atacctatgacgcactgcacatgcaggcectaccacceta g GSGEGRGSLLTCGDVEENPGP T2A peptide (aa) LEGGGEGRGSLLTCGDVEENPGPR T2A peptide (aa) ATNFSLLKOAGDVEENPGP P2A peptide (aa) GSGATNFSLLKQAGDVEENPGP P2A peptide (aa) QCTNYALLKLAGDVESNPGP peptide E2A (aa) GSGQCTNYALLKLAGDVESNPGP peptide E2A (aa) VKOTLNFDLLKLAGDVESNPGP peptide F2A (aa) | 660 - | GSGVKQTLNFDLLKLAGDVESNPGP peptide F2A (aa) Link donor site agtetaaatacages Link donor site teaaciggiatgtos 663 accatctecaaggee Link donor site optimized Link donor site gocecagetita linkage Tacagaga link site: Website 9 link donor cagoacggecagtt aace cgagaac link donor site 999gcgas optimized ctggaaggegagece link donor site
ECT ELI EEE myth 7 ee EEE 671 optimized tattcatataagegg (last 4 nt outside the coding region) Receiver site ge optimized optimized cet optimized ac optimized aac optimized optimized aggagtaagaggagcaggctectgcacagitgactacat atcgetec
RSKRSRLLHSDYMNMTPRRPGPTRKHY aagcggggcagaaagaagctgactcetacatctteaagea sequence of 681 NS. The SAMA ccogaggeagaagasdo cossinalização derived from intracellular cggctgegageta 4-1BB (nt) gatctctggegectacgge human kappa (nt) gatctctagegectataga human kappa (nt) gatctctaggagcatacgga human kappa (nt) gatctecggagcatacgga human kappa (nt) catgcggtgacgtagaggagaatcceggcectagg atgttctgggtgctogtgatogtigacagagtactggectat | Iomínio 688 iegNiggcagagigoiagscia tag gUctaggigcisg transmembrane CD28 tacagcctgctagttacegtagcecttcatcatettttaggte (nd bond donor site cototagataaatt accaagatgaccot site donor connection 9 gaigaceg planned bond donor site tgeactagtaçoado taaactagtaccage bond donor site planned site donor atetecigtaaaas link bond donor site gatoaaggtactois aggacagtaage: Website donor link gaggacagiaagegg link donor site [16% ggteaaggtactoia link donor site tgectoogigtctas link donor site caceaaggigaços! link donor site [6% tgaaciggiatcago atctetigaaatagt link link donor site provided gag site 7 Donor site link gacoagagaaeçois Donor site link tgecagegttagias aatctaagtacagac Donor site link donated Site donor site teaaciggtacatas acaattagtaaggca Donor site link provided ”link accaca ggtatatac donor site provided for connecting bond donor site fecagatosgeca bond donor site cigotciaigagta bond donor site aegcaaagigigtas caacatagtcagttt bond donor site predicted bond donor site 713 aacagaggtgaaaac predicted bond donor site ciggagagiaagesa gaggtgcagctggtgagagtceggaggaggcctagigaa gccaggaggctecetgaggctatetigcgcagecageg gcttcacctttagegactactatatgtcctagatcagacag gcacctggcaagggcctagagtgggtaagctacatcag ctccetetggetecacaatetactatacegactetataaagg gccggtttaccatcagcagagataacgccaagaattece tgtatctgcagatgaacagcctgagggcegaggacaca gccegtgtactattgcgccaaggtagacggcgattacace gaggattattggggccagggcacactagtgaccgtaag 715 ctceggeggeggeggctetagaggaggaggcagegge | scFy BCMA-23 (nt) ggaggaggctcccagtetgecctgacacagecagecag cgtgtccggcteteceggacagtecatcacaatetettgta ceggctetagetecgacgtgggcaagtacaacctagtat cctggtateageagececetggeaaggeccectaagetga tcatctacgatgtgaacaagaggccatctggcgtgagea atcgcttcageggctecaagitctggcaataccegecacact gaccatcagceggectgcagggegacgatgaggcagatt actattgttetagctacggeggcagcagatectacgtatte ggcacaggcaccaaggtgaccgtacta gaggitgcagctggtgcagageggaggaggectagige agcctggcaggtecetacgectatettgcacegecageg 716 gettcacatttggegactatgccatgtectagtteaggeag scFv BCMA-25 (nt) gcaccaggcaagggcctagagtaggatagacttitateege tctaaggcctacggcggcaccacagagtatgcegecag cgtgaagggccaggttcaccatecagcegggacgacteta
[49 agagcatcgcctacetgcagatgaactctetaaagaceg weighed seven aggacacagccgtgtactatigegcagcataggagegec ccaaccgattattagggeccagggcaccctagigacagtg agctceggeggeggeggctetggaggaggaggaageg gaggaggaggatcegacatccagatgacacagtecect goctttetagtecgectetgtaggegatagggtgaccgtgac atgtegegecteccagggceatetetaactaccetggectagt atcagcagaagcceggcaatgececteggetactaatet acagecgcctecacectgcagageggagtgecetecegg ttcagaggaaccggctatggcacagagttttetetgaccat cgacagcctgcagccagaggatttegecacatactattgt cagcagtcttacaccagceggcagacatttggeceeggc acaagactggatatcaag gaggtgcagctagigcagageggaggagacctagitac agcctggcaggtecetaegectatettgcacegecageg gcettcacatttagegactatgccatgtectagttcaageag gcaccaggcaagggcectagagtgggtagactitateege tctaaggcctacggeggcaccacagagtatgcegecag cgtgaagggccggttcaccatcagcegggacgacteta agagcatcgcctacetacagatgaactctetaaagaceg aggacacagccgtgtactatigcgcagcatagagegec ccaaccegattattagggecagggcaccctagigacagtg scFv BCMA-25 (nt) 77 agctecggeggeggeggctetagaggaggaggaageg (O / SSE) gaggaggaggatccegacatccagatgacacagtcececet goct ttetagtecgectetgtaggegatagggtgaccgtgac atgtegegecteccagggceatetetaactacctagectagt atcagcagaagcceggcaatgceceecteggcetactaatet acagcgcctecacectigcagageggagtgcectecogg ttcagaggaaccggctatggcacagagttttetetgaceat cgacagcctgcagccagaggatttegccacatactattgt cagcagtcttacaccagceggcagacatttggececegge acaagactggatatcaag gaggtgcagctagtggagtceggaggaggacctagigaa gccaggaggctcetetgaggctgagectgcgcagectecg gcttcaccttttetgactactatatgagctggatcaggeagg caccaggcaagggcctagagtgggtatettacatcaget 718 cectetggcagcacaatctactatgcegactecgtigaagg scFv BCMA-26 (nt) gcaggttcaccatetetegegataacgecaagaatagec tgtatctgcagatgaactccctgcgggeegaggatacag cegtgtactattacgccaaggtggacggceccecccttecttt gatatctaggggccagggcacaatagtgaccgtgagetec ggaggaggaggatceggeggaggaggctetageggeo
[49 ggcggctcetagetatgtgctgacccagecaccatecgtat semen weighed ctgtggcacctggacagacagcaaggatcacctgtaga gcaaacaatatcggcagcaagtcegtgcactagtacca gcagaagcctaggccaggcecccaatgctagtgatatatga cgatgacgatcggcccageggcatcectgagagattttet ggcagcaactceggcaatacegecacactgaccatetet ggagtggaggcaggcegacgaggcagattacttctgtea cctgtgggacceggagcagagatcactacgtaticggcac aggcaccaagctgaccgtactg gaggtgcagctggtggagtceggaggaggcctagigaa gccaggaggctetetgaggctgagetagcgcagectecg gcettcaccttttetgactactatatgagctggatcaggeagg caccaggcaagggcctagagitgggtatettacatcaget cetetggeagcacaatetactatgcegacteegtigaagg gcaggttcaccatctetegegataacgecaagaatagec tgtatctgcagatgaactecetacgggcegaggatacag cegtgtactattgcgccaaggtagacggcecceccttecttt gatatctggggccagggcacaatggtgaccgtgagetec scFv BCMA-26 (nt) 719 ggaggaggaggatceggeggaggaggctetagegge O / SSE)
ggcggctcetagetatgtgctgacccagecaccatecgtat (ctgtggcacctggacagacagcaaggatcacctgtaga gcaaacaatatcggcagcaagteegtgcactagtacca gcagaagcctagecaggeccecaatgctagtggtatatga cgatgacgatcggcccageggcatecetgagagattttet ggcagcaactccggcaatacegecacactgaccatetet ggagtggaggcaggcgacgaggcagattacttctgtcea cetgtaggacceggagcagagatcactacgtgatteggcac bond donor site aggcaccaagctgacegtacta
720 predicted tcttcatataagegg of truncated marker binding receptor site binding receptor site predicted gge binding receptor site of BCMA-23 if predicted BCMA-25 g predicted BCMA-25 g predicted BCMA-25 g predicted BCMA-25 predicted BCMA-25 predicted to BCMA-52 predicted and BCMA-52 predicted to BCMA-55 predicted receptor binding site 735 binding receptor site ctactatatgtcctagatcagacaggcacctageaaggg predicted BCMA-23 and ETA binding receptor site 736 ggcagattactattgttctagagacggggccggccggcatg predicted BCMA-23 1 hunger ET 737 binding receptor site ctatgccatgtectagtteaageaggcaccaggeaaggg predicted BCMA-25 EA Exma A binding receptor site (O / SSE) binding receptor site 739 agatacagccctagcttteagggccacgtgaccatcage predicted BCMA-52 receptor site> f40 740 cgaggcegattactactgcagcagcaacacceggeca predicted BCMA-55 E Espana E AA binding receptor site (O / SSE)
aee ee binding receptor site is expected from the spacer truncated zeta binding receptor site truncated binding receptor site 748 atggtcagttttetettacagtegteagectgaacataaca | of the truncated marker ASTKGPSVFPLAPCSRSTSESTAALGCLV | KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSNFGTQTYTCNV DHKPSNTKVDKTVERKCCVECPPCPAPP
VAGPSVFLFPPKPKDTLMISRTPEVTCVV 749 VDVSHEDPEVQFNWYVDGVEVHNAKTK | Human IgG2 Fc PREEQFNSTFRVVSVLTVVHQDWLNGKE | (Uniprot PO1859) YKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPS DISVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTOQKSLSLSPGK ASTKGPSVFPLAPCSRSTSESTAALGCLV | KDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTKTYTCNV
DHKPSNTKVDKRVESKYGPPCPSCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCV Human I9G4 Fe 750 VVDVSQEDPEVQFNWYVDGVEVHNAKT (Uniprot PO1861) KPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHE;
497 seven essi [| [Aang The gaggtgcagctagtggagtceggaggaggcctagigaa gccaggaggctecetgaggctatettacgcagecageg gcettcacctttagegactactatatgtcctagatcagacag gcacctggcaagggcctagagigggtgagctacatcag ctccetetggetecacaatetactatacegactetataaagg gcceggtttaccatcagcagagataacgccaagaattece tgtatctgcagatgaacagcctgagggcegaggacaca gccegtgtactattacgccaaggtagacggcgattacace gaggattattggggccagggcacactagtgaccegtaag ctccggeggeggegactetagaggaggaggcagegge ggaggaggctcecagtetgecctgacacagecagecag cgtagtecggcteteceggacagtccatcacaatetettgta ceggctetagetecgacgtgggcaagtacaacctagtat cctggtateagcagececetageaaggeccectaagetga tcatctacgatgtgaacaagaggccatctagegtgagea atcgetteageggetecaagtetaggeaatacegecacact gaccatcagcggcctgcagggegacgatgaggcagatt actattgttctagctacggeggcagcagatcctacgtatte ggcacaggcaccaaggtgaccgtgactagaatctaagta cggaccgccttgtectecttgtecegetectectatigecegg 751 accttccgtgttectattitectecaaagectaaggacacect! Anti-BMCA CAR gatgatcagcaggacccctgaagtgacctacgtagtagt ggatgtgtcccaagaggatcccegaggtacagttcaactg gtatgtagacggcgtggaagtgcacaacgccaagacca | agcctagagaggaacagttccagagcacctacagagto gtgtccegtgctgacagitgctgcaccaggattggctgaacg gcaaagagtacaagtgcaaggtgtecaacaagggect gcctagcagcategagaaaaccatctecaaggecaag ggccagccaagagagccccaggtttacacactgectec aagccaagaggaaatgaccaagaatcaggtgtcccta acatgcctggtcaagggoettetaccectecgatategecgt ggaatgggagagcaatggccagectgagaacaactac aagaccacacctcctgtgctagacagegacggcagttte ttcctgtatagtagactcaccgtagataaatcaagatage aagagggcaacgtgttcagctgcagcgtgatgcacgag gcccetgcacaaccactacacccagaaaagectgagect gtctctaggcaagatgttctaggtactegtagtegtiageg gagtgactagcctattacagcectgctagttacegtggectte atcatcttttgggteaageggggcagaaagaagctgactet acatcttcaagcagcccttcatgcggeeegtgcagacea cacaagaggaagatggctgctectgcagattcecegag
Pa gaagaagaaggcggctacgagetgagagitgaagttca gcagatcegecgacgetecagectatragcagggecaa aaccagctgtacaacgagctgaacctggggagaagag aagagtacgacgtgctggataageggagaggcagaga) tcctgaaataggeggcaageccagacggaagaatecte aagagggcctatataatgagctgcagaaagacaagatg gcegaggcectacagegagateggaatgaagggegagr | gcagaagaggcaagggacacgatggactgtaccagg gcctgagcacegecaceaaggatacctatgacgcacta cacatgcaggccctgccacctaga gaggtgcagctagtgcagageggaggagacctagtac agcctggcaggtecetacgcectatetigcacegecageg gcettcacatttagegactatgccatgtectagttcaageag gcaccaggcaagggcectagagtgggtagactitateege tctaaggcctacggeggcaccacagagtatgcegecag cgtgaagggccggtticaccatcageegggacgacteta agagcatcgcctacetacagatgaactctetaaagaceg aggacacagccgtgtactattgcgcagcatagagegec ccaaccgattattagggeccagggcaccctagigacagtg agcteceggeggeggeggctetagaggaggaggaageg gaggaggaggatcegacatccagatgacacagtecect goctttetatecgectetgtaggegatagggtgaccegtgac atgtegegecteccagggceatetetaactacctagectagt atcagcagaagcceggcaatgececteggcetactaatet acagcgcctecacectgcagageggagtgcectecegg 752 ttcagaggaaccggctatggcacagagttttetetgaccat CAR anti-BMCA cgacagcctgcagccagaggatttegecacatactattgt cagcagtcttacaccagceggcagacatttggececegge acaagactggatatcaaggagtctaaatacggaccgcct | tgtectecttgtecegetectectatigceggaccttecgtatt ccetgatttectecaaagectaaggacaccctgatgatcage aggaccccetgaagtgacctacgtagtagtagatgatatece aagaggatccegaggtgcagttcaactggtatgtagacg gegtggaagtgcacaacgccaagaccaagectagaga ggaacagttccagagcacctacagagtggtgatecgatact gacagtgctgcaccaggattggctgaacggcaaagagt acaagtgcaaggtgtecaacaagggectgcctagcage atcgagaaaaccatctccaaggecaagggecagecaa gagagcccecaggtttacacactgcctecaagecaagag gaaatgaccaagaatcaggtgtccctgacatgcctagte aagggcttctaccecetecgatategeegtagaataggag agcaatggccagcctgagaacaactacaagaccacac
Pa and ctectgtactagacagegacggcagtttettectatatagta gactcaccgtggataaatcaagatggcaagagggcaa cgtgaticagctgcagegtgatgcacgaggceectgcacaa ccactacacccagaaaagectgagectatetetaggcaa gatgttctaggtgctegtagtcgttageggagitgctagect gttacagcctgctagttacegtagecttcatcatettttagat caagcggggcagaaagaagctactetacatcttcaage agcccetteatgcggeecegigcagaccacacaagagga agatggctgctectgcagattcecegaggaagaagaag geggcetgcgagetgagagigaagttcagcagatecgec gacgctccagccetateragcagggccaaaaccagetata caacgagctgaacctggggagaagagaagagtacga cgtgctagataageggagaggcagagatcctgaaatgg gceggcaageccagacggaagaatecteaagagggect gtataatgagctgcagaaagacaagatggccegaggcect acagcgagatcggaatgaagggegagegeagaagag gcaagggacacgatggactgtaccagggcctgageac cgccaccaaggatacctatgacgcactgcacatgcagg cectgecaccetaga gaggtgcagctagtggagtceggaggaggcctagigaa gccaggaggctcetetgaggctgagetacgcagectecg gottcaccttttetgactactatatgagctagatcaggecagg caccaggcaagggcctagagtggatatettacatcaget ccectetggcagcacaatctactatgcegactecgtgaagg gcaggttcaccatetetegegataacgecaagaatagec tgtatctgcagatga actccctgcgggeegaggatacag cegtgatactattgcgccaaggtagacggccecceccttecttt gatatctggggccagggcacaatggtgaccgtgagetec ggaggaggaggatccggeggaggaggctggggggggggggggggggggggggggggggg
753 ctgtggcacctagacagacagcaaggatcacctataga | CAR anti-BMCA gcaaacaatatcggcagcaagtcegtacactagtacca gcagaagcctaggccaggececaatgctagtagtgatatga cgatgacgatcggcccageggcatcectgagagattttct ggcagcaactccggcaatacegecacactgaccatetet ggagtggaggcaggcgacgaggcagattacttctgtca cctgtgggaceggagcagagatcactacgtaticggcac aggcaccaagctgaccgtactggaatcetaagtacggac cgccttgtectecttgtecegetectectatigecggacctte cgtattcctattitectecaaagectaaggacaccctgatga tcagcaggacccctgaagtgacctgcgatagtagtgagatgt gtcccaagaggatccegaggtacagttcaactggtatgtg
[49 sauna weighted gacggcgtagaagtgcacaacgccaagaccaagecta gagaggaacagttccagagcacctacagagtagtatec gtgctgacagitgctgcaccaggattggctgaacggcaaa | gagtacaagtgcaaggtgtecaacaagggcctacctag cagcatcgagaaaaccatctccaaggecaagggecag ccaagagagccccaggtttacacactgcctecaageca agaggaaatgaccaagaatcaggtatecetgacatgcct | About this website gatcaagaggcettetaccectecgatategecgtagaatag gagagcaatggccagcctgagaacaactacaagacca cacctectgtgctagacagegacgagcagtttettectatat agtagactcaccgtggataaatcaagatggcaagaggg caacgtgttcagctgcagegtgatgcacgaggcecctgca caaccactacacccagaaaagectgagectatetetaga caagatgttctaggtactegtagtcgttageggagtgctag cetgttacagcctgctagttacegtggccticatcatetttta ggtcaageggggcagaaagaagctactetacatettcaa | gcagcccttcatgeggeeegtgcagaccacacaagagg aagatggctgctectgcagatteecegaggaagaagaa ggcggctgcgagctgagagtgaagittrcagcagatcege cgacgctcecagectattagcagggeccaaaaccagetat acaacgagctgaacctggggagaagagaagagtacg acgtgctggataageggagaggcagagatcctgaaatg ggcggcaageccagacggaagaatcctraagaggge ctgtataatgagctgcagaaagacaagatggcegagge ctacagcgagatcggaatgaagggegagegeagaag aggcaagggacacgatggactgtaccagggcctgage accgcecaccaaggatacctatgacgcactgcacatgca ggccctgccacctaga agctatgagctgacacagcctecaagegectetageaca | cctggacagcegagtgacaatgagctgtagcggcaccag cagcaacatcggcagccacagegtgaactagtatcage agctgcetagcacageccectaaactgctgatetacacca acaaccagcggcctageggegtgecegatagatttteta gcagcaagagceggcacaagegecagectagcetatttet ggactgcagagegaggacgaggccgactattattgtgacc 754) anti-CAR BMCA gcctgggacggctetetgaacgagcecttatttttagecagagg caccaagctgacagtgctgggatctagaggtageggag gatctggeggeggaggaageggaggeggcggatetett gaaatggctgaagtgcagctggtgcagtctggegeega agtgaagaagcctggegagagectgaagatcagetge aaaggcagcggctacageticaccagetactagategg ctgggtcegacagatgcctageaaaggcctigagtggat
[49 eesetto sauna gggcatcatctaccceggegacagegacaccagataca | gecetagcettteagggccacgtgaccatcagegecgaca agtctatcagcacegectacctgcagitggtecagectgaa ggcctetgacacegecatgtactactgegecagatactet ggcagcttegacaattggggccagggcacactagteac cgtagtccagegagtetaaatacggacegcecttgtectectt gtccegetectectatigceggaccettecgtattcetatttect ccaaagcctaaggacaccctgatgatcageaggacccc | tgaagitgacctgcgtagtggtgagatgtgteccaagaggat ccegaggtacagttcaactagtatgtagacagegtagaa gtgcacaacgccaagaccaagectagagaggaacagt tccagagcacctacagagtggtgtccgtgctgacagitgct gcaccaggattggctgaacggcaaagagtacaagtgc aaggtgtccaacaagggcctacctagcagcategagaa aaccatctecaaggecaagggecagecaagagagecc | caggtttacacactgcctecaagecaagaggaaatgac caagaatcaggtgtccctgacatgcctagtcaagggctte tacccectecgatategecgtagaataggagagcaatage cagcctgagaacaactacaagaccacacctectatacta gacagcgacggcagtttettectatatagtagactcacegt ggataaatcaagatggcaagagggcaacgtgttcagct gcagcgtgatgcacgaggcectgcacaaccactacace cagaaaagcctgagcctgatetetagacaagatgattctagga tactegtggtegttageggagtgctagectattacagecta ctggttaccgtagccttcatcatettttaggteaagegggge agaaagaagctgctctacatcttecaagcageccettcatge ggccegtgcagaccacacaagaggaagatggctgcte ctgcagattcccegaggaagaagaaggcggetacgag ctgagagtgaagttcagcagatcegeegacgetecage ctatcagcagggccaaaaccagctatacaacgagctga acctggggagaagagaagagtacgacgtactggataa gcggagaggcagagatcctgaaatagggeggcaagecc | agacggaagaatccteaagaggagcectatataatgaget gcagaaagacaagatggccegaggcctacagegagatc | ggaatgaagggcgagegcagaagaggcaagggaca cgatggactgtaccagggcctgagcacegecaccaag gatacctatgacgcactgcacatgcaggcccetgecacet 755 ceggacagtetategecatcagetgtaccggcaccaget CAR AGA cagtctgcectgacacagectgccagegttagtgctagte anti-BMCA ctgacgttggctggtatcagcagcacectggcaaggece ctaagctgatgatctacgaggacagcaagaggcecage
[49 semen weighed ggcgtgtecaatagattecageggcageaagageggcaa | cacegecagcctgacaattageggactgcaggeegag gacgaggccgattactactgcagcagcaacacceggte cagcacactggtttttyggeggaggcaccaagetgacagt gctgggatctagaggtageggaggatctggeggeggag gaagcggaggcggceggatctetigaaatggctgaagtg cagctggtgcagtctagegecgagatgaagaaacctgg cgcctetetgaagetgagectgcaaggecageggactacac | cttcategactactacgtgtactagatgcggcaggecect ggacagggactegaatetataggctagatcaaccecaat agcggceggcaccaattacgcccagaaattccagggea gagtgaccatgaccagagacaccagcatcagcacege ctacatggaactgagceggctgagatcegacgacaceg cceatgtactactgegecagatcteagegegacggcetacat ggattattggggccagggaaccctagtcacegigtecag cgagtctaaatacggaccgccttgtectectigtecegete ctcetgttgccggaccttecgtattcetatttectecaaagec taaggacaccctgatgatcagcaggacccctgaagtga cetgegtagtagtagatgatateccaagaggatccegagat | gcagttcaactagtatgtagacggcgtagaagtgcacaa cgccaagaccaagcectagagaggaacagttccagage acctacagagtggtgtecgtactgacagtgctacaccag gattggctgaacggcaaagagtacaagtgcaaggtgte caacaagggcctgcctagcagcatcgagaaaaccatct ccaaggccaagggecagecaagagageccecagattta cacactgcctecaagecaagaggaaatgaccaagaat caggtgtccctgacatgcctagtcaagggcettctaccecte cgatatcgcegtagaataggagagcaatagecagecta agaacaactacaagaccacacctcctgtactggacage gacggcagtttettcctatatagtagactcacegtggataa atcaagatggcaagagggcaacgtgttcagctgcageg tgatgcacgaggccctgcacaaccactacacccagaaa | agcctgagcctatetetaggcaagatgttctgggtactegt gategttageggagtactagectattacagectactagtta cegtggoecttcatcatettttgggteaageggggcagaaa gaagctgctetacatcttcaagcagcecceticatacggece gtgcagaccacacaagaggaagatggctgctectgcag attccecegaggaagaagaaggcggctgacgagetgaga gtgaagttcagcagatcegeegacgcetecagectateag cagggccaaaaccagctgtacaacgagctgaacctag ggagaagagaagagtacgacgtgactggataageggag aggcagagatcctgaaatgggeggcaageccagacgg
[49 sseuenea weighed aagaatcctcaagagggcctatataatgagctgcagaa agacaagatggccegaggcectacagegagateggaatg aagggcgagegcagaagaggcaagggacacgatgg actgtaccagggcctgagcacegecaccaaggatacct atgacgcactgcacatgcaggccectagccaccetaga cagtctgccctgacacagectgecagegttagtactagte ceggacagtetategecatcagetgtaccggcaccaget ctgacgttggctggtatcagcagcaccctggeaaggece ctaagctgatgatctacgaggacagcaagaggcecage ggcgtgtecaatagatticageggcagcaagageggcaa cacegecagcectgacaattageggactgcaggeegag gacgaggccgattactactgcagcagcaacacceggte cagcacactggtttttyggcggaggcaccaagetgacagt getgggatctagaggtageggaggatctageggeggag gaagcggaggcggceggatctetigaaatggctgaagta cagctggtgcagtctggegecgagatgaagaaacctgg cgccetetetgaagetgagetgcaaggecageggctacac; cttcategactactacgtgtactagatgcggcaggeccect ggacagggactcgaatctatgggctggatcaaccccaat agcggceggcaccaattacgcccagaaattccagggea gagitgaccatgaccagagggacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgacgggg
756 ggattattagggccagagaaccctagtcacegtgtecag | CAR anti-BMCA cgagtctaaatacggacecgcecttgtectectigtecegete ctectgttgceggaccttecagtattectatitectecaaages taaggacaccctgatgatcagcaggacccctgaagtga ccetgegtaggtagtggatgtateccaagaggatccegagagt gcagttcaactagtatatagacggcgtagaagigcacaa cgccaagaccaagcectagagaggaacagttccagage acctacagagtggtgtcegtgctgacagtgctacaccag gattggctgaacggcaaagagtacaagtgcaaggtgte caacaagggcctgcctagcagcategagaaaaccatet ccaaggccaagggecagecaagagagececagattta cacactgcctecaagecaagaggaaatgaccaagaat caggtgtccctgacatgcctagtcaagggcettctacceecte cgatatcgcegtiggaataggagagcaatggccagectg agaacaactacaagaccacacctcctgtactggacage gacggcagtttettcctatatagtagactcacegtggataa atcaagatggcaagagggcaacgtgattcagctgcageg tgatgcacgaggccctgcacaaccactacacccagaaa | agcctgagcctatetetaggcaagatgttctaggtactegt me and gategttageggagtactagectattacagcctgactagtta cegtggecttcatcatettttagggteaggagtaagaggagr | aggctcectgcacagigactacatgaacatgactcccege cgcecegggeccaceogcaageattaccagecetatge cccaccacgegacttegcagectategetecagagtigaa gttcagcagatcegecgacgcetecagectatrageaggg ccaaaaccagctgtacaacgagctgaacciggggaga agagaagagtacgacgtgctggataageggagaggca gagatcctgaaatgggeggcaageccagacggaaga atccteaagagggcectatataatgagetacagaaagac aagatggcegaggcctacagegagateggaatgaagg gegagegcagaagaggcaagggacacgatggactat accagggcctgagcacegecaceaaggatacctatgac; gcactgcacatgcaggccctgccacctaga EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGDYTEDYWGQGT LVTVSSGGGGSGGGGSGGGGSASALTOA | PASVSGSPGQSITISCTGSSSDVGKYNLV SWYQQPPGKAPKLIIYDVNKRPSGVSNR FSGSKSGNTATLTISGLOGDDEADYYCS SYGGSRSYVFGTGTKVTVLESKYGPPCP PCPAPPVAGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFQOSTYRVVSVLTVLHQD: 157 WLNGKEYKCKVSNKGLPSSIEKTISKAKG CAR anti-BMCA QPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGKMFWVLV VVGGVLACYSLLVTVAFIIFWVKRGRKKL LYIFKQPFMRPVQTTAQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAY QQGQONAL YNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR
EVOLVASGGGLVQPGRSLRLSCTASGFT FGDYAMSWFKQAPGKGLEWVGFIRSKA; 758 | yeeTttEYAASVKGRFTISRDDsKslaYLQ | Anti-BMCA CAR MNSLKTEDTAVYYCAAWSAPTDYWGQG
[57 | seeuêne the eesgiving TLVIVSSGGGESCCCESCCGESDIAMT QSPAFLSASVGDRVTVTCRASQGISNYL
AWYQQKPGNAPRLLIYSASTLOSGVPSR FRGTGYGTEFSLTIDSLQPEDFATYYCQA | SYTSROTFGPGTRLDIKESKYGPPCPPC PAPPVAGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFQOSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGKMFWVLVVWV GGVLACYSLLVTVAFIIFWVKRGRKKLLY! FKQPFMRPVOTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDAL HMOQALPPR EVOLVESGGGLVKPGGSLRLSCAASGFT FSDYYMSWIRQAPGKGLEWVSYISSSGS TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCAKVDGPPSFDIWGQGTM VTVSSGGGESCEGESCEGESSYVLTAP | PSVSVAPGQTARITCGANNIGSKSVHWY QQKPGQAPMLVVYDDDDRPSGIPERFS GSNSGNTATLTISGVEAGDEADYFCHLW DRSRDHYVFGTGTKLTVLESKYGPPCPP
CPAPPVAGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVH | 759 NAKTKPREEQFOSTYRVvsvLTVLHaDWw | Anti-BMCA CAR LNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCS | VMHEALHNHYTOKSLSLSLGKMFWVLVV VGGVLACYSLLVTVAFIIFWVKRGRKKLL YIFKQPFMRPVOTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNALY NELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGM
[57 | seBuêA o umesemão
KGERRRGKGHDGLYQGLSTATKDTYDAL and
SYELTQPPSASGTPGQRVTMSCSGTSSN IGSHSVNWYQQLPGTAPKLLIVTNNQRPS | GVPDRFSGSKSGTSASLAISGLOSEDEA DYYCAAWDGSLNGLVFGGGTKLTVLGS ADDITIONAL INFORMATION SGAEVKKPGESLKISCKGSGYSFTSYWIG WVRQMPGKGLEWMGIIYPGDSDTRYSP SFQGHVTISADKSISTAYLQWSSLKASDT AMYYCARYSGSFDNWGQGTLVTVSSES KYGPPCPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFQSTYRVVSV | 760) TvLHQDWLNGKEYKCKVSNKGLPssIE | Anti-BMCA CAR KTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTOQKSLSLSL GKMFWVLVVVGGVLACYSLLVTVAFIIFW VKRGRKKLLYIFKQPFMRPVQTTQEEDG CSCRFPEEEEGGCELRVKFSRSADAPAY QQGANQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTI ATKDTYDALHMQALPPR QSALTQPASVSASPGOQSIAISCTGTSSDV GWYQQHPGKAPKLMIYEDSKRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYC SSNTRSSTLVFGGGTKLTVLGSRGGGGS GEGESCEGESLEMAEVOALVOSGAEMK KPGASLKLSCKASGYTFIDYYVYWMRQA
PGQOGLESMGWINPNSGGTNYAQKFOGR VTMTRDTSISTAYMELSRLRSDDTAMYY: 761 CARSQRDGYMDYWGOGTLVTVSsEsKy | Anti-BMCA CAR GPPCPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFOSTYRVVSVLT VLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWOE
[49 jeesetto sauna GNVFSCSVMHEALHNHYTOKSLSLSLGK MFWVLVVVGGVLACYSLLVTVAFIIFWVK
RGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQ | GQONQLYNELNLGRREEYDVLDKRRGRD
PEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTAT | KDTYDALHMQALPPR QSALTQPASVSASPGQSIAISCTGTSSDV GWYQQHPGKAPKLMIYEDSKRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYC SSNTRSSTLVFGGGTKLTVLGSRGGGGS GGGGSCGGGGSLEMAEVOQLVOSGAEMK KPGASLKLSCKASGYTFIDYYVYWMRQA PGQAOGLESMGWINPNSGGTNYAQKFOGR VTMTRDTSISTAYMELSRLRSDDTAMYY CARSQRDGYMDYWGQGTLVTVSSESKY GPPCPPCPAPPVAGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFQSTYRVVSVLT: 762 VLHQDWLNGKEYKCKVSNKGLPSSIEKTI CAR anti-BMCA SKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTOKSLSLSLGK MFWVLVVVGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRD
PEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTAT |
KDTYDALHMQALPPR binding receptor site binding receptor site
67th | sseuemeia eee o [redicted “=== == ac predicted LPVLTQPPSTSGTPGQRVTVSCSGSSSN! I GSNVVFWYQQLPGTAPKLVIVRNNQRPS GVPDRFSVSKSGTSASLAISGLRSEDEAD
YYCAAWDDSLSGYVFGTGTKVTVLGSR GECESCEGESCGEGESLEMAEVOLVOS: 769 GAEVKKPGSSVKVSCKASGGTFSSYAIS | Anti-BMCA SeFv WVRQAPGOGLEWMGRIIPILGIANYAQKF QGRVTMTEDTSTDTAYMELSSLRSEDTA
VYYCARSGYSKSIVSYMDYWGQGTLVTV ss QAVLTQPPSASGTPGQRVTISCSGSSSNI GSNYVFWYQQLPGTAPKLLIYSNNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEA
DYYCAAWDDSLSASYVFGTGTKVTVLGS REGESECEGESCEGESLEMAQVALVA: 17th SGAEVKKPGSSVKVSCKASGGTFssYal | Anti-BCMA SCFV SWVRQAPGAGLEWMGRIIPILGTANYAQ KFQGRVTITADESTSTAYMELSSLRSEDT
AVYYCARSGYGSYRWEDSWGQGTLVTV ss QSVLTQPPSVSGAPGORVTISCTGSSSNI GAGFDVHWYQQLPGTAPKLLIVGNSNRP SGVPDRFSGSKSGTSASLAITGLOAEDE
ADYYCQSYDSSLSGYVFGTGTKVTVLGS REGESECEGESCEGESLEMAQVALVA: To SGAEVKKPGASVKVSCKASGYTFTDYYM | Anti-BCMA SºFV HWVRQAPGORLEWMGWINPNSGGTNY AQKFQDRITVTRDTSSNTGYMELTRLRS
DDTAVYYCARSPYSGVLDKWGQGTLVT vss EVQLVOSGAEVKKPGSSVKVSCKASGGT |
FSSYAISWVRQAPGQGLEWMGRIIPILGI 772 - | ANVAQKFOGRVTMTEDTSTDTAYMELSS | Anti-BMCA VH LRSEDTAVYYCARSGYSKSIVSYMDYWG OGTLVTVSS
QVQLVOSGAEVKKPGSSVKVSCKASGG 773 - | TESSYAISWVRQAPGQOGLEWMGRIIPILG | Anti-BMCA VH TANYAQKFQGRVTITADESTSTAYMELSS
Women and LRSEDTAVYYCARSGYGSYRWEDSWGA | eme QVQLVOSGAEVKKPGASVKVSCKASGYT]
FTDYYMHWVRQAPGQRLEWMGWINPN 774 | SGGTNYAQKFQDRITVTRDTSSNTGYME | Anti-BMCA VH LTRLRSDDTAVYYCARSPYSGVLDKWGQ | GTLVTVSS
LPVLTQPPSTSGTPGQRVTVSCSGSSSNI GSNVVFWYQQLPGTAPKLVIVRNNQRPS: 1 GVPDRFSVSKSGTSASLAISGLRSEDEAD | Anti-BMCA VI YYCAAWDDSLSGYVFGTGTKVTVLG
QAVLTQPPSASGTPGQRVTISCSGSSSNI GSNYVFWYQQLPGTAPKLLIYSNNQRPS: 176 GVPDRFSGSKSGTSASLAISGLRSEDEA | Anti-BMCA V DYYCAAWDDSLSASYVFGTGTKVTVLG
QSVLTQPPSVSGAPGQRVTISCTGSSSNI GAGFDVHWYQQLPGTAPKLLIYGNSNRP: 7 SGVPDRFSGSKSGTSASLAITGLOAEDE | Anti-BMCA V ADYYCOSYDSSLSGYVFGTGTKVTVLG LPVLTQPPSASGTPGORVTISCSGRSSNI GSNSVNWYRQLPGAAPKLLIVSNNQRPP GVPVRFSGSKSGTSASLAISGLQSEDEAT
YYCATWDDNLNVHYVFGTGTKVTVLGSR GECESCEGESCEGESLEMAQVALVAS | 78 GAEVKKPGSSVKVSCKASGGTFSSYAIs | Anti-BCMA SCFV WVRQAPGQOGLEWMGRIIPILGIANYAQKF QGRVTITADKSTSTAYMELSSLRSEDTAV
YYCARGGYYSHDMWSEDWGOQGTLVTV ss EVOLLESGGGLVQPGGSLRLSCAASGFT FSSYAMSWVRQAPGKGLEWVSAISGSG GSTYYADSVKGRFTISRDNSKNTLYLQM
NSLRAEDTAVYYCARAEMGAVFDIWGQ 782 | GTMVTVSSGSTSGSGKPGSGEGSTKGEI | anti-BCMA scFv VLTOSPATLSLSPGERATLSCRASQOSVS RYLAWYQQKPGQAPRLLIYDASNRATGI! PARFSGSGSGTDFTLTISSLEPEDFAVYY
CQQRISWPFTFGGGTKVEIK name e EIVLTOSPATLSLSPGERATLSCRASQOSV SRYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYY
CQQRISWPFTFGGGTKVEIKRGSTSGSG 783 KPGSGEGSTKGEVQLLESGGGLVQPGG | scFv anti-> BCMA SLRLSCAASGFTFSSYAMSWVRQAPGK GLEWVSAISGSGGSTYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARA EMGAVFDIWGQGTMVTVSS QVQLVESGGGVVQPGRSLRLSCAASGF TFSSYGMHWVRQAPGKGLEWVAVISYD GSNKYYADSVKGRFTISRDNSKNTLYLO
MNSLRAEDTAVYYCARDGTYLGGLWYF DLWGRGTLVTVSSGSTSGSGKPGSGEG: 784 STKGDIVMTQSPLSLPVTPGEPASISCRsS | Anti-BCMA SºFv SQOSLLHSNGYNYLDWYLQKPGQOSPALLI YLGSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMOQGLGLPLTFGGGTK VEIK DIVMTQOSPLSLPVTPGEPASISCRSSOSL LHSNGYNYLDWYLQKPGQSPOQLLIYLGS NRASGVPDRFSGSGSGTDFTLKISRVEA
EDVGVYYCMQGLGLPLTFGGGTKVEIKR GSTSGSGKPGSGEGSTKGQVALVESGG | 785 GVVQPGRSLRLSCAASGFTFSSYGMHW | Anti-BCMA SCFV
VRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAV |
YYCARDGTYLGGLWYFDLWGRGTLVTV ss QVQLVOSGAEVKKPGASVKVSCKASGYT] FTSYYMHWVRQAPGQGLEWMGIINPGG GSTSYAQKFOGRVTMTRDTSTSTVYMEL
SSLRSEDTAVYYCARESWPMDVWGQGT 786 TVTVSSGSTSGSGKPGSGEGSTKGEIVM | anti-BCMA scFv
TQSPATLSVSPGERATLSCRASQSVSSN LAWYQQKPGQAPRLLIYGASTRATGIPAR | FSGSGSGTEFTLTISSLOSEDFAVYYCQQ
YAAYPTFGGGTKVEIK EIVMTQOSPATLSVSPGERATLSCRASQSV | SSNLAWYQQKPGQAPRLLIVGASTRATG! : 787 PARFSGSGSGTEFTLTISSLASEDFAVYY | Anti-BCMA SeFv CQQYAAYPTFGGGTKVEIKRGSTSGSGK
RPE if eee PGSGEGSTKGQVQLVOSGAEVKKPGAS VKVSCKASGYTFTSYYMHWVRQAPGQOG LEWMGIINPGGGSTSYAQKFQGRVTMTR DTSTSTVYMELSSLRSEDTAVYYCARES
WPMDVWGQGTTVTVSS QLQALQESGPGLVKPSETLSLTCTVSGGSI | SSSSYYWGWIRQPPGKGLEWIGSISYSG STYYNPSLKSRVTISVDTSKNQFSLKLSS
VTAADTAVYYCARGRGYATSLAFDIWGQ 788 GTMVTVSSGSTSGSGKPGSGEGSTKGEI | anti-BCMA scFv VLTQSPATLSLSPGERATLSCRASQSVSS | YLAWYQQKPGQAPRLLIYDASNRATGIPA | RFSGSGSGTDFTLTISSLEPEDFAVYYCQ QRHVWPPTFGGGTKVEIK EIVLTASPATLSLSPGERATLSCRASQSV SSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYY
CQAQARHVWPPTFGGGTKVEIKRGSTSGS 789 GKPGSGEGSTKGQLQLQESGPGLVKPS | anti-BCMA scFv ETLSLTCTVSGGSISSSSYYWGWIRQPP GKGLEWIGSISYSGSTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARG RGYATSLAFDIWGQGTMVTVSS EVOLVESGGGLVQPGGSLRLSCAASGFT FSSYSMNWVRQAPGKGLEWVSTISSSSS) TIYYADSVKGRFTISRDNAKNSLYLQMNS
LRAEDTAVYYCARGSQEHLIFDYWGQOGT 790 LVTVSSGSTSGSGKPGSGEGSTKGEIVL | anti-BCMA scFv TQSPATLSLSPGERATLSCRASQSVSRY LAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQ QRFYYPWTFGGGTKVEIK EIVLTASPATLSLSPGERATLSCRASQSV
SRYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYY |
CQAOQRFYYPWTFGGGTKVEIKRGSTSGS 791 GKPGSGEGSTKGEVQLVESGGGLVQPG | anti-BCMA scFv
GSLRLSCAASGFTFSSYSMNWVRQAPG KGLEWVSTISSSSSTIYYADSVKGRFTISR | DNAKNSLYLQMNSLRAEDTAVYYCARGS
QEHLIFDYWGQGTLVTVSS ese fes TFSSYGMAWVRQAPGKGLEWVAVISYD GSNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARTDFWSGSPPGL DYWGQGTLVTVSSGSTSGSGKPGSGEG STKGDIQLTQSPSSVSASVGDRVTITCRA SOGISSWLAWYQQKPGKAPKLLIYGASS LASGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQIYTFPETFGGGTKVEIK DIQLTASPSSVSASVGDRVTITCRASQG! SSWLAWYQQKPGKAPKLLIVGASSLOSG VPSRFSGSGSGTDFTLTISSLQPEDFATY
YCQAIYTFPFTFGGGTKVEIKRGSTSGSG 793 - | KPGSGEGSTKGQVOQLVESGGGVVOPGR | anti-BCMA scFv SLRLSCAASGFTFSSYGMHWVRQAPGK GLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARTD FWSGSPPGLDYWGQGTLVTVSS QVOLVOSGAEVKKPGSSVKVSCKASGG TFSSYAISWVRQAPGOGLEWMGGIIPIFG TANYAQKFQGRVTITADESTSTAYMELSS
LRSEDTAVYYCARTPEYSSSIWHYYYGM DVWGOGTTVTVSSGSTSGSGKPGSGEG: TOA STKGDIVMTQSPDSLAVSLGERATINCKs | Anti-BCMA SeFv SQOSVLYSSNNKNYLAWYQQKPGQPPKLL IYWASTRESGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQFAHTPFTFGGGTKV EIK DIVMTOSPDSLAVSLGERATINCKSSOSV LYSSNNKNYLAWYQQKPGQPPKLLIVWA STRESGVPDRFSGSGSGTDFTLTISSLOA
EDVAVYYCOQFAHTPFTFGGGTKVEIKR GSTSGSGKPGSGEGSTKGQVAQLVOSGA | 7º5 EVKKPGSSVKVSCKASGGTFSSYAISwv | Anti-BCMA SºFV RQOAPGQGLEWMGGIIPIFGTANYAQKFQ GRVTITADESTSTAYMELSSLRSEDTAVY YCARTPEYSSSIWHYYYGMDVWGQGTT VTVSS QVOLVESGGGVVOPGRSLRLSCAASGF
TFSSYGMHWVRQAPGKGLEWVAVISYD 796 - | GSNKYYADSVKGRFTISRDNSKNTLYLQ | anti scFv:> BCMA MNSLRAEDTAVYYCVKGPLQEPPYDYG MDVWGQGTTVTVSSGSTSGSGKPGSGE
[in the straight semen | GSTKGEIVMTQSPATLSVSPGERATLSC RASQSVSSNLAWYQQKPGQAPRLLIYSA STRATGIPARFSGSGSGTEFTLTISSLOS EDFAVYYCQQHHVWPLTFGGGTKVEIK EIVMTQOSPATLSVSPGERATLSCRASQSV SSNLAWYQQKPGOAPRLLIYSASTRATG! PARFSGSGSGTEFTLTISSLOSEDFAVYY
CQQHHVWPLTFGGGTKVEIKRGSTSGS 797 GKPGSGEGSTKGQVOLVESGGGVVOPG | anti-BCMA scFv RSLRLSCAASGFTFSSYGMHWVRQAPG KGLEWVAVISYDGSNKYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCVKG PLQEPPYDYGMDVWGOQGTTVTVSS DIVLTOSPASLAVSLGERATINCRASESV SVIGAHLIHWYQQKPGQPPKLLIYLASNL ETGVPARFSGSGSGTDFTLTISSLOAEDA
AIYYCLOSRIFPRTFGQGTKLEIKGSTSGS 798 GKPGSGEGSTKGQVQLVOSGSELKKPG | anti-BCMA scFv ASVKVSCKASGYTFTDYSINWVRQAPGQ GLEWMGWINTETREPAYAYDFRGRFVFS LDTSVSTAYLQISSLKAEDTAVYYCARDY SYAMDYWGQGTLVTVSS DIVLTOSPASLAVSLGERATINCRASESV SVIGAHLIHWYQQKPGQPPKLLIYLASNL ETGVPARFSGSGSGTDFTLTISSLOAEDA
AIYYCLOSRIFPRTFGOGTKLEIKGSTSGS 799 GKPGSGEGSTKGQVQLVOSGSELKKPG | anti-BCMA scFv ASVKVSCKASGYTFTDYSINWVRQAPGQ GLEWMGWINTETREPAYAYDFRGRFVFS LDTSVSTAYLQISSLKAEDTAVYYCARDY SYAMDYWGQOGTLVTVSS DIVLTOSPASLAVSLGERATINCRASESV SVIGAHLIHWYQQKPGQPPKLLIYLASNL ETGVPARFSGSGSGTDFTLTISSLQAEDA
AIYYCLOSRIFPRTFGOGTKLEIKGSTSGS 800 GKPGSGEGSTKGQVQLVOSGSELKKPG | anti-BCMA scFv ASVKVSCKASGYTFTDYSINWVRQAPGQ GLEWMGWINTETREPAYAYDFRGRFVFS LDTSVSTAYLQISSLKAEDTAVYYCARDY SYAMDYWGQGTLVTVSS EVOLVESGGGLVQPGGSLRLSCAVSGFA:
FR ee STYYAASVKGRFTISRDNSRNTLYLQMN SLRPEDTAIYYCSAHGGESDVWGQGTTV TVSSASGGGGSGGRASGGGGSDIALTA SPSSLSASVGDRVTITCRASQSISSYLNW YQQKPGKAPKLLIVAASSLOSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQOQSY STPYTFGQGTKVEIK QVQLVESGGGLVQPGRSLRLSCAASGFT FSNYAMSWVRQAPGKGLGWVSGISRSG ENTYYADSVKGRFTISRDNSKNTLYLQM
NSLRDEDTAVYYCARSPAHYYGGMDVW 802 - | GAGTTVTIVSSASGGGGSGGRASGGGG | anti scFv:> BCMA SDIVLTOSPGTLSLSPGERATLSCRASOS ISSSFLAWYQQKPGQAPRLLIVGASRRAT GIPDRFSGSGSGTDFTLTISRLEPEDSAV YYCQQYHSSPSWTFGQGTKLEIK QVQLVESGGGLVQPGGSLRLSCAVSGF ALSNHGMSWVRRAPGKGLEWVSGIVYS GSTYYAASVKGRFTISRDNSRNTLYLQM
NSLRPEDTAIYYCSAHGGESDVWGQGTT 803 - | VIVSSASGGGGSGGRASGGGGSDIRLT | anti-BCMA scFv QSPSPLSASVGDRVTITCQASEDINKFLN WYHQTPGKAPKLLIYDASTLQTGVPSRF SGSGSGTDFTLTINSLQPEDIGTYYCQQY ESLPLTFGGGTKVEIK EVOLVESGGGLVQPGGSLRLSCAVSGFA LSNHGMSWVRRAPGKGLEWVSGIVYSG STYYAASVKGRFTISRDNSRNTLYLQMN
SLRPEDTAIYYCSAHGGESDVWGQGTTV 804 - | TVSSASGGGGSGGRASGGGGGSEIVLTQ | scFv anti-> BCMA SPGTLSLSPGERATLSCRASOSIGSSSLA WYQQAKPGAQAPRLLMYGASSRASGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQ YAGSPPFTFGQGTKVEIK QIQLVAOSGPELKKPGETVKISCKASGYTF TDYSINWVKRAPGKGLKWMGWINTETRE
PAYAYDFRGRFAFSLETSASTAYLQINNL KYEDTATYFCALDYSYAMDYWGQGTSV: 805 TVSSGGEESCEGESCEGESDIVLTOSP | Anti-BCMA SFv ASLAMSLGKRATISCRASESVSVIGAHLIH WYQQKPGQPPKLLIVLASNLETGVPARF
SGSGSGTDFTLTIDPVEEDDVAIYSCLOS no semen o eeetie [LRFBRIFGGOTREER O QIQALVASGPELKKPGETVKISCKASGYTF RHYSMNWVKQAPGKGLKWMGRINTESG VPIYADDFKGRFAFSVETSASTAYLVINNL
KDEDTASYFCSNDYLYSLDFWGQGTALT 806 VSSGGGGSGGGGSGGGGSDIVLTOSPP | scFv anti- <BCMA SLAMSLGKRATISCRASESVTILGSHLIYW
YQQKPGQPPTLLIQLASNVQTGVPARFS GSGSRTDFTLTIDPVEEDDVAVYYCLOSR | TIPRTFGGGTKLEIK QIQALVASGPELKKPGETVKISCKASGYTF THYSMNWVKQAPGKGLKWMGRINTETG EPLYADDFKGRFAFSLETSASTAYLVINN
LKNEDTATFFCSNDYLYSCDYWGQGTTL 807 TVSSGGGGSGGGGSGGGGSDIVLTOSP | anti-BCMA scFv PSLAMSLGKRATISCRASESVTILGSHLIY WYQQKPGQPPTLLIQLASNVQTGVPARF SGSGSRTDFTLTIDPVEEDDVAVYYCLOS RTIPRTFGGGTKLEIK QVALVASGAEVKKPGASVKVSCKASGY SFPDYYINWVRQAPGQGLEWMGWIYFA SGNSEYNQKFTGRVTMTRDTSINTAYME
LSSLTSEDTAVYFCASLYDYDWYFDVWG 808 QGTMVTVSSGCGGGSGGGGSGGGGSDI | anti-BCMA scFv VMTOTPLSLSVTPGQPASISCKSSOSLVH SNGNTYLHWYLQKPGQOSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAED VGIYYCSQOSSIYPWTFGQGTKLEIK QVALVASGAEVKKPGASVKVSCKASGY SFPDYYINWVRQAPGQGLEWMGWIYFA SGNSEYNQKFTGRVTMTRDTSINTAYME
LSSLTSEDTAVYFCASLYDYDWYFDVWG 809 QGTMVTVSSGGGGSGGGGSGGGGSDI | anti-BCMA scFv VMTQTPLSLSVTPGQPASISCKSSQOSLVH SNGNTYLHWYLQKPGQOSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAED VGIYYCSQOSSIYPWTFGQAGTKLEIK QVALVASGAEVKKPGASVKVSCKASGY
SFPDYYINWVYRQAPGQGLEWMGWIYFA 810 SGNSEYNQKFTGRVTMTRDTSINTAYME | anti-BCMA scFv LSSLTSEDTAVYFCASLYDYDWYFDVWG QGTMVTVSSGGGGSGGGGSGGGGSDI
RPE love fee VMTQTPLSLSVTPGQPASISCKSSOSLVH SNGNTYLHWYLQKPGQOSPQLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAED VGIYYCSQSSIYPWTFGQGTKLEIK QVALVAOSGAEVKKPGASVKVSCKASGY SFPDYYINWVRQAPGOGLEWMGWIYFA SGNSEYNQKFTGRVTMTRDTSSSTAYM
ELSSLRSEDTAVYFCASLYDYDWYFDVW 811 GAGTMVTVSSGGGGSGGGGSGGGGSD | anti-BCMA scFv IVMTQOTPLSLSVTPGEPASISCKSSOSLV HSNGNTYLHWYLQKPGQSPQLLIYKVSN RFSGVPDRFSGSGSGADFTLKISRVEAE DVGVYYCAETSHVPWTFGQGTKLEIK QVALVAOSGAEVKKPGASVKVSCKASGY SFPDYYINWVRQAPGQGLEWMGWIYFA SGNSEYNQKFTGRVTMTRDTSSSTAYM
ELSSLRSEDTAVYFCASLYDYDWYFDVW 812 GAGTMVTVSSGGGGSGGGGSGGGGSD | anti-BCMA scFv IVMTQTPLSLSVTPGEPASISCKSSOSLV HSNGNTYLHWYLQKPGQSPQLLIYKVSN RFSGVPDRFSGSGSGADFTLKISRVEAE DVGVYYCAETSHVPWTFGQGTKLEIK QVAQLVOSGAEVKKPGASVKVSCKASGY SFPDYYINWVRQAPGQGLEWMGWIYFA SGNSEYNQKFTGRVTMTRDTSSSTAYM
ELSSLRSEDTAVYFCASLYDYDWYFDVW 813 GAGTMVTVSSGCGGGSGGGGSGGGGSD | anti-BCMA scFv IVMTQTPLSLSVTPGEPASISCKSSOSLV HSNGNTYLHWYLQKPGQSPQLLIYKVSN RFSGVPDRFSGSGSGADFTLKISRVEAE
DVGVYYCAETSHVPWTFGQGTKLEIK QVALVESGGGLVOQPGGSLRLSCEASGFT |
LDYYAIGWFRQAPGKEREGVICISRSDGS 814 TYYADSVKGRFTISRDNAKKTVYLQMISL | anti-BCMA Vx KPEDTAAYYCAAGADCSGYLRDYEFRG QGTAVTVSS QVALVASGAEVKKPGSSVKVSCKASGG
TFSSYAISWVRQAPGQGLEWMGRIIPILGI 815 ANYAQKFOGRVTITADKSTSTAYMELSSL | Anti-BMCA VH RSEDTAVYYCARGGYYSHDMWSEDWG QGTLVTVSS
Res me EVOLLESGGGLVOQPGGSLRLSCAASGFT
FSSYAMSWVRQAPGKGLEWVSAISGSG 816 GSTYYADSVKGRFTISRDNSKNTLYLQM | Anti-BMCA VH NSLRAEDTAVYYCARAEMGAVFDIWGQ GTMVTVSS QVALVESGGGVVQAQPGRSLRLSCAASGF
TFSSYGMHWVRQAPGKGLEWVAVISYD 817 GSNKYYADSVKGRFTISRDNSKNTLYLQ | Anti-BMCA VH MNSLRAEDTAVYYCARDGTYLGGLWYF
DLWGRGTLVTVSS QVALVOSGAEVKKPGASVKVSCKASGYT] |
FTSYYMHWVRQAPGQGLEWMGIINPGG 818 GSTSYAQKFOGRVTMTRDTSTSTVYMEL | Anti-BMCA VH SSLRSEDTAVYYCARESWPMDVWGQOGT
TVTVSS QLALQESGPGLVKPSETLSLTCTVSGGS |
SSSSYYWGWIRQPPGKGLEWIGSISYSG 819 STYYNPSLKSRVTISVDTSKNQFSLKLSS | Anti-BMCA VH VTAADTAVYYCARGRGYATSLAFDIWGQ GTMVTVSS
EVOLVESGGGLVQPGGSLRLSCAASGFT FSSYSMNWVRQAPGKGLEWVSTISSSSS | 820 TIYYADSVKGRFTISRDNAKNSLYLQMNS | Anti-BMCA VH LRAEDTAVYYCARGSQEHLIFDYWGQOGT LVTVSS QVALVESGGGVVQPGRSLRLSCAASGF
TFSSYGMHWVRQAPGKGLEWVAVISYD 821 GSNKYYADSVKGRFTISRDNSKNTLYLQ | Anti-BMCA VH MNSLRAEDTAVYYCARTDFWSGSPPGL DYWGQGTLVTVSS QVALVOSGAEVKKPGSSVKVSCKASGG
TFSSYAISWVRQAPGQGLEWMGGIIPIFG 822 TANYAQKFQGRVTITADESTSTAYMELSS | Anti-BMCA VH LRSEDTAVYYCARTPEYSSSIWHYYYGM DVWGQGTTVTVSS QVALVESGGGVVQPGRSLRLSCAASGF
TFSSYGMHWVRQAPGKGLEWVAVISYD 823 GSNKYYADSVKGRFTISRDNSKNTLYLOQ | Anti-BMCA VH MNSLRAEDTAVYYCVKGPLQEPPYDYG MDVWGQGTTVTVSS
[Re love fee FTDYSINWVRQAPGQGLEWMGWINTET REPAYAYDFRGRFVFSLDTSVSTAYLQAIS SLKAEDTAVYYCARDYSYAMDYWGQOGT LVTVSS EVOLVESGGGLVQPGGSLRLSCAVSGFA)
LSNHGMSWVRRAPGKGLEWVSGIVYSG 825 STYYAASVKGRFTISRDNSRNTLYLQMN | Anti-BMCA VH SLRPEDTAIY YCSAHGGESDVWGQGTTV
TVSS QVALVESGGGLVOPGRSLRLSCAASGFT |
FSNYAMSWVRQAPGKGLGWVSGISRSG 826 ENTYYADSVKGRFTISRDNSKNTLYLQM | VH anti- <BMCA NSLRDEDTAVYYCARSPAHYYGGMDVW GQGTTVTVSS QVQALVESGGGLVQPGGSLRLSCAVSGF
ALSNHGMSWVRRAPGKGLEWVSGIVYS 827 GSTYYAASVKGRFTISRDNSRNTLYLQM | Anti-BMCA VH NSLRPEDTAIY YCSAHGGESDVWGQGTT] VTVSS EVOLVESGGGLVQPGGSLRLSCAVSGFA,)
LSNHGMSWVRRAPGKGLEWVSGIVYSG 828 STYYAASVKGRFTISRDNSRNTLYLQMN | Anti-BMCA VH SLRPEDTAIY YCSAHGGESDVWGOGTTV TVSS QIQALVASGPELKKPGETVKISCKASGYTF
RHYSMNWVKQAPGKGLKWMGRINTESG 829 VPIYADDFKGRFAFSVETSASTAYLVINNL | Anti-BMCA VH
KDEDTASYFCSNDYLYSLDFWGQGTALT vsSs QIQALVASGPELKKPGETVKISCKASGYTF
THYSMNWVKQAPGKGLKWMGRINTETG 830 EPLYADDFKGRFAFSLETSASTAYLVINN | VH anti- <BMCA LKNEDTATFFCSNDYLYSCDYWGQGTTL TVSS QVALVOSGAEVKKPGASVKVSCKASGY
SFPDYYINWVRQAPGQGLEWMGWIYFA 831 SGNSEYNQKFTGRVTMTRDTSINTAYME | Anti-BMCA VH LSSLTSEDTAVYFCASLYDYDWYFDVWG QGTMVTVSS QVALVASGAEVKKPGASVKVSCKASGY; SGNSEYNQKFTGRVTMTRDTSSSTAYM ELSSLRSEDTAVYFCASLYDYDWYFDVW GOGTMVTVSS
LPVLTQPPSASGTPGQRVTISCSGRSSNI GSNSVNWYRQLPGAAPKLLIVYSNNQRPP: 833 GVPVRFSGSKSGTSASLAISGLASEDEAT | Y | anti-BMCA YYCATWDDNLNVHYVFGTGTKVTVLG
EIVLTOSPATLSLSPGERATLSCRASQSV SRYLAWYQQKPGQAPRLLIVYDASNRATG: 8% IPARFSGSGSGTDFTLTISSLEPEDFAvyy | YL anti-BMCA CQQRISWPFTFGGGTKVEIK
DIVMTQSPLSLPVTPGEPASISCRSSQSL LHSNGYNYLDWYLQKPGOSPOQLLIYLGS: 835 NRASGVPDRFSGSGSGTDFTLKISRVEA | V | anti-BMCA
EDVGVYYCMQGLGLPLTFGGGTKVEIK EIVMTQSPATLSVSPGERATLSCRASQSV | SSNLAWYQQKPGQAPRLLIVGASTRATGI: 836 PARFSGSGSGTEFTLTISSLASEDFAVYYy | V- anti-BMCA CQQYAAYPTFGGGTKVEIK
EIVLTQSPATLSLSPGERATLSCRASQSV SSYLAWYQQKPGQAPRLLIYDASNRATGI: ss PARFSGSGSGTDFTLTISSLEPEDFAVYY | Y | anti-BMCA CQAQRHVWPPTFGGGTKVEIK
EIVLTOSPATLSLSPGERATLSCRASQOSV SRYLAWYQQKPGQAPRLLIVDASNRATG | 838 - | pARESGSGSGTDFTLTISSLEPEDFAvYY | Y | antiBMCA
CQOQRFYYPWTFGGGTKVEIK DIQLTASPSSVSASVGDRVTITCRASQG! SSWLAWYQQKPGKAPKLLIYGASSLOSG | 839 VPSRFSGSGSGTDFTLTISSLOPEDFATY | Anti-BMCA YE YCQAQIYTFPETFGGGTKVEIK
DIVMTQSPDSLAVSLGERATINCKSSQSV LYSSNNKNYLAWYQQKPGQPPKLLIYWA: 840) sTRESGVPDRFSGSGSGTDFTLTISSLOA | Y | antiBMCA
EDVAVYYCQQFAHTPFTFGGGTKVEIK EIVMTQOSPATLSVSPGERATLSCRASQSV | SSNLAWYQQKPGQAPRLLIVSASTRATGI: sa PARFSGSGSGTEFTLTISSLASEDFAVYy | YE anti-BMCA
CQQHHVWPLTFGGGTKVEIK DIVLTASPASLAVSLGERATINCRASESV: SVIGAHLIFAWYQQKPGAPPKLLIVLASNL | Anti-BMCA VE ETGVPARFSGSGSGTDFTLTISSLQAEDA AIYYCLOSRIFPRTFGQGTKLEIK
DIQLTASPSSLSASVGDRVTITCRASQSIS SYLNWYQQKPGKAPKLLIYAASSLOSGV: sas PSRFSGSGSGTDFTLTISSLQPEDFATYY | Y | anti-BMCA
CQQSYSTPYTFGOGTKVEIK DIVLTASPGTLSLSPGERATLSCRASOQS! SSSFLAWYQQKPGQAPRLLIVGASRRAT: sa GIPDRFSGSGSGTDFTLTISRLEPEDSAV | Anti-BMCA YE YYCQOQYHSSPSWTFGQGTKLEIK
DIRLTOSPSPLSASVGDRVTITCQASEDIN KFLNWYHQTPGKAPKLLIYDASTLOTGVP: sas SRESGSGSGTDFTLTINSLOPEDIGTYYC | Anti-BMCA VL
QQYESLPLTFGGGTKVEIK EIVLTOSPGTLSLSPGERATLSCRASOS! GSSSLAWYQQKPGQAPRLLMYGASSRA: sas SGIPDRFSGSGSGTDFTLTISRLEPEDFA | Y) anti-BMCA VYYCQAYAGSPPFTFGOGTKVEIK
DIVLTASPPSLAMSLGKRATISCRASESV TILGSHLIVWYQQKPGQPPTLLIQLASNV: sa QTGVPARFSGSGSRTDFTLTIDPVEEDD | Anti-BMCA VI VAVYYCLOSRTIPRTFGGGTKLEIK
DIVMTQTPLSLSVTPGQPASISCKSSQSL VHSNGNTYLHWYLQKPGQSPOQLLIYKVS: sas NRFSGVPDRFSGSGSGTDFTLKISRVEA | V | anti-BMCA EDVGIYYCSQSSIYPWTFGQGTKLEIK
DIVMTQTPLSLSVTPGEPASISCKSSQSL VHSNGNTYLHWYLQKPGQSPOQLLIYKVS: are NRFSGVPDRFSGSGSGADFTLKISRVEA | Anti-BMCA VI
EDVGVYYCAETSHVPWTFGQGTKLEIK 850 atgcttctectagtgacaagoccttcetactetataagttacca | chain signal sequence cacccagcattcctectgatecca alpha from GMCSFR peptide chain signal 851 MLLLVTSLLLCELPHPAFLLIP Sta from GMCSFR Link receptor site 854 aagtttctttetgtatticcagactgacegtggataaatcte optimized 855 GAGTCTAAATACTGGCCGGCCGGC Spacer - optimized by CCTTGTCCAGCTCCTCCTGTTGCCGGA | codon (nt)
[49 seen the eesetto CCTTCCEGTGTTCCTGTTTCCTCCAAAGC CTAAGGACACCCTGATGATCAGCAGGA →TGAAGTGACCTGCGTGGTGGTE GATGTGTCCCAAGAGGATCCCGAGGTG CAGTTCAATTGGTACGTGGACGGCGTG GAAGTGCACAACGCCAAGACCAAGCCT AGAGAGGAACAGTTCCAGAGCACCTAC AGAGTGGTGTCCGTGCTGACAGTGCTG CACCAGGATTGGCTGAACGGCAAAGAG TACAAGTGCAAGGTGTCCAACAAGGGC CTGCCTAGCAGCATCGAGAAAACCATC TCCAAGGCCAAGGGCCAGCCAAGAGA GCCCCAGGTTTACACACTGCCTCCAAG CCAAGAGGAAATGACCAAGAATCAGGT GTCCCTGACATGCCTGGTCAAGGGCTT
CTACCCCTCCGATATCGCCGTGGAATG GGAGAGCAATGGCCAGCCTGAGAACAA | CTACAAGACCACACCTCCTGTGCTGGA CAGCGACGGCAGTTTCTTCCTGTATAG TAGACTCACCGTGGATAAATCAAGATG GCAAGAGGGCAACGTGTTCAGCTGCAG CGTGATGCACGAGGCCCTGCACAACCA CTACACCCAGAAAAGCCTGAGCCTGTC
TCTGGGCAAA gaatctaagtacggaccgccttgtectectigtecegetect cetgttgceggaccttecgtattcetatitectecaaagecta aggacaccctgatgatcagcaggacccectgaagtgace tagcgtggtagtagatgtateccaagaggatccegaggta cagttcaactggtatgtagacggcgatagaagtgcacaac gccaagaccaagcctagagaggaacagttccagagea cctacagagtggtgtecgtactgacagtgctacaccagg attggctgaacggcaaagagtacaagtgcaaggtatec 856 aacaagggcctgcetagcagcategagaaaaccatete | CO / SSE spacer caaggccaagggccagecaagagagcecccaggtttac | Alternative (nt) acactgcctecaagecaagaggaaatgaccaagaate aggtgtccctgacatgcctggteaagggcettetaceeetes gatatcgcegtagaatagggagagcaatggccagectga gaacaactacaagaccacacctcctatactagacageg acggcagtttcttectatatagtagactcaccgtagataaat caagatggcaagagggcaacgtgattcagctacagegta atgcacgaggccctgcacaaccactacacccagaaaa gcctgagcctgtetetaggcaag

权利要求:
Claims (275)
[1]
1. Chimeric antigen receptor characterized by the fact that it comprises: (a) an extracellular antigen binding domain that specifically recognizes the B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.
[2]
2. Chimeric antigen receptor, according to claim 1, characterized by the fact that the spacer comprises a portion of an immunoglobulin constant region.
[3]
3. Chimeric antigen receptor, according to claim 1 or 2, characterized by the fact that the spacer comprises a sequence of a joint region, a Cn2 region and a Cr3 region.
[4]
4. Chimeric antigen receptor, according to claim 3, characterized by the fact that the articulation region comprises all or a portion of an IgG4 articulation region and / or an IgG2 articulation region, in which the I9G4 hinge region is optionally a human IgG4 hinge region, and the IgG2 hinge region is optionally a human IgG2 hinge region; the CH2 region comprises all or a portion of an IgG4 CH2 region and / or an IgG2 CH2 region, wherein the IgG4 CH2 region is optionally a human IgG4 CH2 region, and the CH2 region IgG2 is optionally a CH2 region of human IgG2; and / or the CH3 region comprises all or a portion of an IgG4 CH3 region and / or an I9gG2 CH3 region, wherein the IgG4 CH3 region is optionally a human IgG4 CH3 region, and CH3 region of IgG2 is optionally a region of
Human IgG2 CH3.
[5]
5. Chimeric antigen receptor, according to claim 3 or 4, characterized by the fact that the joint, Ch2 and CH3 comprises all or a portion of each of an IgG4 joint region, Cx2 and Ch3.
[6]
6. Chimeric antigen receptor, according to claim 3 or 4, characterized by the fact that: the Cn2 region is chimeric and comprises a Crn2 region of human IgG4 and human I9G2; the Cn2 region is chimeric and comprises a CH2 region of human IgG4 and human IgG2; and / or the CH3 region is chimeric and comprises a CH3 region of human IgG4 and human IgG2.
[7]
Chimeric antigen receptor according to any one of claims 1 to 6, characterized in that the spacer comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint comprising at least one substitution amino acid substitution compared to the human I9G4 joint region; a chimeric human I9G2 / 4 Ch2 region; and a human IgG4 Ch3 region.
[8]
Chimeric antigen receptor according to any one of claims 1 to 4, 6 and 7, characterized in that the spacer is or comprises (i) the sequence mentioned in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity for SEQ ID NO: 649; or (iii) a contiguous portion of (i) or (ii) with at least 125 amino acids in length.
[9]
Chimeric antigen receptor according to any one of claims 1 to 3 and 6 to 8, characterized in that the spacer is or comprises the sequence mentioned in SEQ ID NO:
649.
[10]
10. Chimeric antigen receptor characterized by the fact that it comprises: (a) an extracellular antigen binding domain that specifically recognizes the B cell maturation antigen (BCMA); (b) a spacer mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[11]
11. Chimeric antigen receptor according to any one of claims 1 to 10, characterized by the fact that the antigen binding domain is an antibody fragment comprising a variable heavy chain (Vx) and a region of variable variable light chain (V.).
[12]
12. Chimeric antigen receptor, according to claim 11, characterized by the fact that: the Vx region is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in any of SEQ ID NOs: 617, 115, 256, 519 or 609; and the V. region is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity sequence for the Vi region amino acid sequence mentioned in any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[13]
13. Chimeric antigen receptor, according to claim 11 or 12, characterized by the fact that: the V "region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively te, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 617 and 618, respectively;
the V region "and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 256 and 267, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 256 and 267, respectively, the Vx region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 519 and 535, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 519 and 535 , respectively, the Vx region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 115 and 536, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 115 and 536, respectively; or the Vx region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 609 and 610, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 609 and 610, respectively.
[14]
14. Chimeric antigen receptor according to any one of claims 11 to 13, characterized by the fact that the Vy region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 617 and 618, respectively.
[15]
15. Chimeric antigen receptor according to any one of claims 11 to 13, characterized in that: the Va region comprises a heavy chain complementarity determination region 1 (CDR-H1), a de - heavy chain complementarity termination 2 (CDR-H2) and a heavy chain complementarity determination region 3 (CDR-H3) contained within the amino acid sequence of the Vu region selected from any of SEQ ID NOs: 617, 115 , 256, 519 or 609; and the V. region comprises a light chain complementarity determination region 1 (CDR-L1), a light chain complementarity determination region 2 (CDR-L2) and a chain complementarity determination region take 3 (CDR-L3) contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[16]
16. Chimeric antigen receptor according to any one of claims 11 to 15, characterized by the fact that: the Vu region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of Vu region mentioned in SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained in the amino acid sequence of region V. mentioned in SEQ ID NO: 618.
[17]
17. Chimeric antigen receptor, according to any one of claims 11 to 13 and 15, characterized by the fact that: the Vx region comprises (a) a CDR-H1 comprising the selected amino acid sequence from any of the SEQ ID NOs: 1, 2, 507 or 593; (b) a CDR-H2 comprising the selected amino acid sequence from any of SEQ ID NOs: 4, 5, 513 or 594; and (c) a CDR-H3 comprising the selected amino acid sequence from any of SEQ ID NOs: 7, 10, 157, 517 or 595; and the V. region comprises (a) a CDR-L1 comprising the selected amino acid sequence from any of SEQ ID NOs: 33, 178, 380, 589 or 601; (b) a CDR-L2 comprising the selected amino acid sequence from any of SEQ ID NOs: 43, 183, 400, 590 or 602; and (c) a CDR-L3 comprising the selected amino acid sequence from any of SEQ | D
NOs: 194, 416, 421, 591 or 603.
[18]
18. Chimeric antigen receptor according to any one of claims 11 to 13, 15 and 17, characterized by the fact that: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the sequence of amino acids of SEQ ID NOS: 593, 594 and 595, respectively, and the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 2, 5 and 157, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the amino acid sequence of SEQ ID NOS: 178, 183 and 194, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 1,4 and 7, respectively, and the Vi region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the amino acid sequence of SEQ ID NOS: 380, 400 and 416, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 2, 5 and 10, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 33, 43 and 421, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 507, 513 and 517, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 589, 590 and 591, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615 and 603, respectively.
[19]
19. Chimeric antigen receptor according to any one of claims 11 to 18, characterized in that a Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively.
[20]
20. Chimeric antigen receptor according to any one of claims 11 to 13, 15, 17 and 18, characterized in that a Vu region is or comprises the amino acid sequence mentioned in any of the SEQ IDs NOs: 617, 115, 256, 519 or 609; and a V region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[21]
21. The chimeric antigen receptor according to any one of claims 11 to 13, 15, 17, 18 and 20,
characterized by the fact that: the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617; and the V region is or comprises the amino acid sequence mentioned in SEQ ID NO: 618 the VH region is or comprises the amino acid sequence mentioned in SEQ ID NO: 256; 22z and a V region is or comprises the sequence amino acid mentioned in SEQ ID NO: 267; the VH region is or comprises the amino acid sequence mentioned in SEQ ID NO: 519; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 535; the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 115; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 536; or the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 609; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 610.
[22]
22. Chimeric antigen receptor according to any one of claims 11 to 21, characterized in that: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 593, 594 and 595, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 611, 612 and 613, respectively; and the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602 and 603, respectively; or the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 614, 615 and 603, respectively.
[23]
23. Chimeric antigen receptor according to any one of claims 11 to 22, characterized in that the V4 region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 618.
[24]
24. Chimeric antigen receptor according to any one of claims 11 to 23, characterized in that the fragment comprises an scFv.
[25]
25. Chimeric antigen receptor according to any one of claims 11 to 24, characterized by the fact that, when the Vu region, and the V. region, are joined by a flexible ligand.
[26]
26. Chimeric antigen receptor, according to claim 25, characterized by the fact that scFv comprises a linker that comprises the amino acid sequence GGGGSCGGGGSGGGGS (SEQ ID NO: 361).
[27]
27. Chimeric antigen receptor according to any one of claims 11 to 26, characterized by the fact that the Vu region is amino-terminal to the V ..
[28]
28. Chimeric antigen receptor according to any of claims 11 to 27, characterized in that the antigen-binding domain comprises the amino acid sequence selected from any of SEQ ID NOs: 478, 128 -139, 268-278, 329, 442, 558- 576, 578-583, 585 or 769-771 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the selected amino acid sequence of any of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585 or 769-
771.
[29]
29. Chimeric antigen receptor according to any one of claims 1 to 28, characterized by the fact that the antigen binding domain comprises the amino acid sequence selected from any of SEQ ID NOs: 478, 278 , 559,
560 or 442 or an amino acid sequence having sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the selected amino acid sequence of any of SEQ ID NOs: 478, 278, 559, 560 or 442.
[30]
30. Chimeric antigen receptor according to any one of claims 1 to 29, characterized in that the antigen-binding domain comprises the amino acid sequence mentioned in SEQ ID NO: 478 or an amino acid sequence - acids that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the amino acid sequence mentioned in SEQ ID NO: 478.
[31]
31. Chimeric antigen receptor according to any one of claims 1 to 30, characterized in that the antigen-binding domain comprises the amino acid sequence mentioned in SEQ ID NO: 478.
[32]
32. Chimeric antigen receptor according to any one of claims 1 to 31, characterized in that a nucleic acid encoding the antigen-binding domain comprises (a) the nucleotide sequence mentioned in any one SEQ ID NOS: 648, 352, 647, 716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity to any of SEQ ID NOS: 648, 352, 647, 716 or 718; or (c) a degenerate sequence of (a) or (b).
[33]
33. Chimeric antigen receptor according to any one of claims 1 to 32, characterized in that the nucleic acid encoding the antigen-binding domain comprises the nucleotide sequence mentioned in any of SEQ ID NO : 460, 440, 715, 717 or 719.
[34]
34. The chimeric antigen receptor according to any one of claims 1 to 33, characterized in that the nucleic acid encoding the antigen-binding domain comprises the nucleotide sequence mentioned in SEQ ID NO: 460 .
[35]
35. Chimeric antigen receptor according to any one of claims 11 to 26, characterized by the fact that the Vy region is a carboxy-terminal to the V ..
[36]
36. Chimeric antigen receptor, characterized by the fact that it comprises: (1) an extracellular antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA), in which the extracellular antigen-binding domain with - comprises: (i) a variable heavy chain (Va) comprising an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in SEQ ID NO: 617; and (ii) a variable light chain (V.) region comprising an amino acid sequence having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the V region amino acid sequence mentioned in any of SEQ ID NO: 618; (2) a spacer comprising an I9gG4 / 2 chimeric joint or a modified I9G4 joint; an I9G2 / 4 chimeric Cn2 region; and an IgG4 Ch3 region, optionally about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region that comprises a cytoplasmic signaling domain of a CD3-zeta chain
(CD3C) and an intracellular signaling domain of a T cell costimulatory molecule
[37]
37. Chimeric antigen receptor, according to claim 36, characterized by the fact that: the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vu region mentioned in SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. mentioned in SEQ ID NO: 618; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615 and 603, respectively.
[38]
38. Chimeric antigen receptor, characterized by the fact that it comprises:
(1) an extracellular antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA), in which the extracellular antigen-binding domain comprises:
a variable heavy region (Vx4) comprising CDR-H1, CDR-H2 and CDR-H3 contained within the Vx region amino acid sequence mentioned in SEQ ID NO: 617; and a light variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 contained within the V region amino acid sequence mentioned in SEQ ID NO: 618; or a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and a V. region comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively;
a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and a V. region comprising CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively;
a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and a V region, comprising CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or a Vx region comprising CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and a Vi region. comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence
acids of SEQ ID NOS: 614, 615 and 603, respectively; (2) a spacer that comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint; an IgG2 / 4 chimeric CrH2 region; and a CH3 region of IgG4, optionally about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD37) chain and an intracellular signaling domain of a T cell costimulatory molecule.
[39]
39. Chimeric antigen receptor according to any one of claims 36 to 38, characterized in that the extracellular antigen binding domain comprises the Vx region amino acid sequence mentioned in SEQ ID NO: 617, and the amino acid sequence of region V. mentioned in SEQ ID NO: 618
[40]
40. Chimeric antigen receptor according to any one of claims 1 to 35, characterized by the fact that the intracellular signaling region comprises an activation cytoplasmic signaling domain.
[41]
41. Chimeric antigen receptor, according to claim 40, characterized by the fact that the activation cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, it is a component of T cell receptor (TCR) and / or comprises an immunoreceptor tyrosine-based activation motif (ITAM).
[42]
42. Chimeric antigen receptor, according to claim 40 or 41, characterized by the fact that the signal domain
Cytoplasmic activation is or comprises a cytoplasmic signaling domain of a CD3-zeta (CD36) chain or a functional variant or signaling portion thereof.
[43]
43. The chimeric antigen receptor according to any one of claims 40 to 42, characterized by the fact that the cytoplasmic activation domain is human or is a human protein.
[44]
44. The chimeric antigen receptor according to any one of claims 36 to 39, 42 and 43, characterized by the fact that the cytoplasmic signaling domain is or comprises the sequence mentioned in SEQ ID NO: 628 or a sequence of amino acids that have at least 90% sequence identity to SEQ ID NO: 628.
[45]
45. Chimeric antigen receptor according to any one of claims 40 to 44, characterized by the fact that the intracellular signaling region further comprises a co-stimulating signaling region.
[46]
46. Chimeric antigen receptor, according to claim 45, characterized by the fact that the co-stimulatory signaling region comprises an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof.
[47]
47. Chimeric antigen receptor according to any one of claims 36 to 39, 45 and 46, characterized by the fact that the co-stimulating signal region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or signaling portion thereof.
[48]
48. Chimeric antigen receptor according to any one of claims 36 to 39 and 45 to 47, characterized by the fact that the co-stimulating signaling region comprises a domain
4-1BB intracellular signaling.
[49]
49. Chimeric antigen receptor according to any one of claims 36 to 39 and 45 to 48, characterized by the fact that the co-stimulating signal region is human or is a human protein.
[50]
50. Chimeric antigen receptor according to any one of claims 36 to 39 and 45 to 49, characterized in that the co-stimulating signal region is or comprises the sequence mentioned in SEQ ID NO: 626 or a amino acid sequence that exhibits at least 90% sequence identity to the sequence mentioned in SEQ ID NO: 626.
[51]
51. Chimeric antigen receptor according to any one of claims 36 to 50, characterized by the fact that the co-stimulatory signaling region is between the trans-membrane domain and the intracellular signaling region.
[52]
52. The chimeric antigen receptor according to any one of claims 1 to 51, characterized in that the transmembrane domain is or comprises a CD4, CD28 or CD8 transmembrane domain.
[53]
53. Chimeric antigen receptor, according to claim 52, characterized by the fact that the transmembrane domain is or comprises a transmembrane domain of a CD28.
[54]
54. The chimeric antigen receptor according to any one of claims 1 to 53, characterized by the fact that the transmembrane domain is human or that of a human protein.
[55]
55. Chimeric antigen receptor according to any one of claims 1 to 54, characterized in that the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 624 or an amino acid sequence that exhibits at least 90% sequence identity for SEQ ID NO: 624.
[56]
56. The chimeric antigen receptor according to any one of claims 1 to 55, characterized by the fact that the chimeric antigen receptor comprises its N to C terminal in order: the antigen binding domain, the spacer, the transmembrane domain, and the intracellular signaling domain.
[57]
57. Chimeric antigen receptor according to any one of claims 1 to 56, characterized by the fact that (a) the ability of the antigen-binding domain or the chimeric antigen receptor to bind to BCMA expressed on the surface of a target cell, or (b) a measure indicative of chimeric antigen receptor function or activity after exposure of cells that express the chimeric antigen receptor to cells that express surface BCMA, is not reduced or blocked or is not substantially reduced or blocked in the presence of a concentration or amount of a soluble or spillable form of BCMA, wherein the concentration or amount is a concentration or amount capable of blocking or reducing or substantially blocking or reduce binding or a measure of function or activity associated with a reference anti-BCMA recombinant receptor or a reference anti-BCMA binding domain, under the same or substantially the same co conditions, or is a concentration or quantity present in a biological sample.
[58]
58. Chimeric antigen receptor, according to claim 57, characterized by the fact that the concentration or amount of the soluble or spillable form of BCMA: is a concentration or quantity present in the individual's serum or blood or plasma or of a patient with multiple myeloma or an average concentration or amount present in the serum, blood or plasma of patients within a population of patients with multiple myeloma or a subtype or subpopulation thereof, or is a concentration or quantity at which the binding or measurement is reduced or blocked, or substantially reduced or blocked, to a reference anti-BCMA recombinant receptor, optionally a reference anti-BCMA CAR, under the same conditions or substantially the same conditions.
[59]
59. The chimeric antigen receptor according to any one of claims 1 to 58, characterized in that the chimeric antigen receptor is encoded by a poly-nucleotide sequence comprising the sequence mentioned in any of SEQ ID NOS: 751-756 or by a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99% sequence identity for the sequence mentioned in any of SEQ ID NOS: 751-756.
[60]
60. Chimeric antigen receptor according to any one of claims 1 to 59, characterized in that the chimeric antigen receptor is encoded by a polylucleotide sequence comprising the sequence mentioned in any of SEQ ID NOS : 755 and 756 or by a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96% , 97%, 98% or 99% sequence identity for the sequence mentioned in any of SEQ ID NOS: 755 and 756.
[61]
61. The chimeric antigen receptor according to any one of claims 1 to 60, characterized in that the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence mentioned in SEQ ID NO: 755 or by a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in this.
[62]
62. The chimeric antigen receptor according to any one of claims 1 to 61, characterized in that the chimeric antigen receptor is encoded by a polylucleotide sequence comprising the sequence mentioned in SEQ ID NO: 755 .
[63]
63. Polynucleotide characterized by the fact that it encodes the chimeric antigen receptor as defined in any one of claims 1 to 62.
[64]
64. Polynucleotide, according to claim 63, characterized by the fact that after expression of the polynucleotide in a human cell, optionally a human T cell, the RNA, optionally the messenger RNA (mMRNA), of the polynucleotide, it exhibits at least 70%, 75%, 80%, 85%, 90% or 95% RNA homogeneity.
[65]
65. Polynucleotide according to claim 63 or 64, characterized in that the encoded chimeric antigen receptor comprises a spacer comprising a chimeric IgG4 / 2 joint or a modified I9G4 joint; an I9G2 / 4 chimeric Ch2 region; and an IgG4 CH3 region, optionally about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649, a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity for SEQ ID NO: 649.
[66]
66. Polynucleotide encoding a chimeric antigen receptor, characterized by the fact that it comprises the nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, where after expression of the polynucleotide in a human cell, optionally a human T cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide, exhibits at least 70%, 75%,
80%, 85%, 90% or 95% RNA homogeneity.
[67]
67. Polynucleotide according to any one of claims 63 to 66, characterized by the fact that the coded spacer comprises a portion of an immunoglobulin.
[68]
68. Polynucleotide, according to any of claims 63 to 67, characterized by the fact that the coded spacer comprises a sequence of a joint region, a Ch2 region and a Cn3 region.
[69]
69. Polynucleotide, according to any one of claims 63, 64 and 66 to 68, characterized by the fact that the articulation region comprises all or a portion of an IgG4 articulation region and / or an articulation region IgG2, where the I9gG4 hinge region is optionally a human I9G4 hinge region, and the IgG2 hinge region is optionally a human IgG2 hinge region; the CH2 region comprises all or a portion of an IgG4 CH2 region and / or an IgG2 Chn2 region, wherein the IgG4 Ch2 region is optionally a human IgG4 Cx2 region, and the Ch2 region IgG2 is optionally a Cn2 region of human IgG2; and / or the CH3 region comprises all or a portion of an IgG4 CH3 region and / or an IgG2 CH3 region, wherein the IgG4 CH3 region is optionally a human IgG4 CH3 region, and CH3 region of IgG2 is optionally a CH3 region of human IgG2.
[70]
70. Polynucleotide, according to any of claims 63, 64 and 66 to 69, characterized by the fact that the joint, Ch2 and Ch43 comprises all or a portion of each of a joint region, Ch2 and CH3 IgG4.
[71]
71. Polynucleotide, according to any one of the
vindications 63, 64 and 66 to 69, characterized by the fact that: the articulation region is chimeric and comprises an articulation region of human IgG4 and human IgG2; the Cn2 region is chimeric and comprises a Cn2 region of human IgG4 and human I9gG2; and / or the CH3 region is chimeric and comprises a CH3 region of human IgG4 and human IgG2.
[72]
72. Polynucleotide, according to any of claims 63, 64 and 66 to 71, characterized by the fact that the spacer comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint comprising at least an amino acid substitution compared to the human IgG4 hinge region; a human I9G2 / 4 chimeric Cx2 region; and a Ch43 region of human IgG4.
[73]
73. Polynucleotide, according to any of claims 63, 64 and 66 to 72, characterized by the fact that the encoded spacer is or comprises (i) the sequence mentioned in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO: 649 that has at least 95%, 96%, 97%, 98% or 99% of sequence identity for SEQ ID NO: 649; or (iii) a contiguous portion of (i) or (ii) with at least 125 amino acids in length.
[74]
74. Polynucleotide, according to any of claims 63 to 73, characterized by the fact that the coded spacer is or comprises the sequence mentioned in SEQ ID NO:
649.
[75]
75. Polynucleotide according to any one of claims 63 to 74, characterized by the fact that the nucleic acid encoding the spacer comprises at least one modified donor and / or binding receptor site, said donor site and / or modified binding receptor comprising one or more nucleotide modifications corresponding to a reference binding donor site and / or reference binding receptor site contained in the sequence mentioned in SEQ ID NO: 621.
[76]
76. Polynucleotide according to claim 75, characterized in that the one or more nucleotide modifications comprise a nucleotide substitution.
[77]
77. Polynucleotide according to claim 75 or 76, characterized by the fact that reference binding donor sites and / or reference binding receptors are canonical, non-canonical or cryptic binding sites.
[78]
78. Polynucleotide, according to any of claims 75 to 77, characterized by the fact that: the reference binding donor site (s) and / or binding receptor (s) reference site has (have) a binding site prediction score of at least or about 0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0, 90, 0.95, 0.99 or 1.0; and / or the referral link donor site (s) and / or referral link receiver (s) is / are expected to be involved in a liaison event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[79]
79. Polynucleotide according to any of claims 75 to 78, characterized by the fact that: the donor site of the reference link comprises the sequence aatctaagtacggac (SEQ ID NO: 705), traactagtacatag (SEQ ID NO: 706), acaattagtaaggca (SEQ ID NO: 707) and / or accacaggtatatac (SEQ ID NO: 708); and / or the reference binding receptor site comprises the sequence aagtttctttetatattecaggcetgacegtggataaatete (SEQ ID NO: 742) and / or gaggcaacdgtattctettgcagtgteatgcacgaagecetge (SEQ ID NO: 743).
[80]
80. Polynucleotide, according to any one of the
vindications 75 to 78, characterized by the fact that: the referral donor site (s) and / or referral link recipient (s) have a prediction score from the at least or about 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99 or 1.0; and / or the referral donor site (s) and / or referral link receiver (s) is / are expected to be involved in a liaison event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[81]
81. Polynucleotide, according to any of claims 75 to 78 and 80, characterized by the fact that: the donor site of the reference link comprises the tcaactagtacgtag sequence (SEQ ID NO: 706); and / or the reference binding receptor site comprises the sequence aagtttctttetatattecaggetgacegtggataaatete (SEQ ID NO: 742).
[82]
82. Polynucleotide according to any of claims 75 to 81, characterized by the fact that at least one of the one or more nucleotide modifications is within 1, 2,3, 4,5,6,7,8 , 9 or 10 residues from the junction of the reference binding site and / or reference binding donor site.
[83]
83. Polynucleotide according to any of claims 75 to 82, characterized by the fact that one or more nucleotide changes are silent and / or result in a degenerate codon compared to SEQ ID NO: 621 and / or it does not change the amino acid sequence of the coded spacer.
[84]
84. Polynucleotide according to any of claims 75 to 83, characterized by the fact that: the modified donor binding site is mentioned in agtttataatacggac (SEQ ID NO: 661), teaactagtatatag (SEQ ID NO: 662 ), accatctccaaggec (SEQ ID NO: 663) and / or gceccecaggtttacac (SEQ ID NO:
664); and / or the modified binding receptor site is mentioned in ca- gtttcttectatatagtagactcacegtggataaatcaa (SEQ ID NO: 672), gggcaaco- tgttcagctgcagegtgatgcacgaggecetgc (SEQ ID NO: 673) and / or cgccttg- tec
[85]
85. Polynucleotide according to any one of claims 75 to 84, characterized by the fact that the modified donor binding site is mentioned in tcaactggtatatag (SEQ ID NO: 662) and / or the modified receptor site is mentioned in cagtttcttectata - tagtagactagaccagcatagataaatcaa (SEQ ID NO: 672) and / or cgaccttg- tectecttatecegetectectattaceggacet (SEQ ID NO: 766).
[86]
86. Polynucleotide, according to any of claims 63 to 85, characterized by the fact that the spacer is codified by the nucleotide sequence mentioned in SEQ ID NO: 622 or in a portion thereof.
[87]
87. Polynucleotide encoding a chimeric antigen receptor, characterized by the fact that the polynucleotide comprises the nucleic acid that encodes: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is or comprises the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649; (c) a transmembrane domain; and (d) an intracellular signaling region.
[88]
88. Polynucleotide encoding a chimeric antigen receptor, characterized by the fact that the polynucleotide comprises nucleic acid that encodes: (a) an extracellular antigen binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid essentially consists or consists of the sequence mentioned in SEQ ID NO: 622 or encodes an amino acid sequence mentioned in SEQ ID NO: 649;
(c) a transmembrane domain; and (d) an intracellular signaling region.
[89]
89. Polynucleotide according to claim 87 or 88, characterized by the fact that, after the expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (MRNA), of the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90% or 95% RNA homogeneity.
[90]
90. Polynucleotide, according to any of claims 63 to 89, characterized by the fact that, after expression in a human cell, optionally a human T cell, the transcribed RNA, optionally messenger RNA (mMRNA), of the polynucleotide - deo exhibits reduced heterogeneity compared to the heterogeneity of the mRNA transcribed from a reference polynucleotide, said reference polynucleotide that encodes the same amino acid sequence as the polynucleotide, in which the reference polynucleotide differs in the presence of one or more binding donor sites and / or one or more binding receptor sites on the nucleic acid encoding the spacer and / or comprises one or more nucleotide modifications compared to the polynucleotide and / or comprises the sequence mentioned in SEQ ID NO: 621.
[91]
91. Polynucleotide according to claim 90, characterized by the fact that the heterogeneity of RNA is reduced by more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more.
[92]
92. Polynucleotide according to claim 90 or 91, characterized by the fact that the transcribed RNA, optionally messenger RNA (mMRNA), of the reference polynucleotide exhibits more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity.
[93]
93. Polynucleotide, according to any one of the
vindications 63 to 92, characterized by the fact that RNA homogeneity and / or heterogeneity is / are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation or chromatography liquid.
[94]
94. Polynucleotide, according to any of claims 63 to 93, characterized by the fact that the polynucleotide is codon-optimized for expression in a human cell.
[95]
95. Polynucleotide, according to any of claims 63 to 94, characterized by the fact that the antigen is associated with a disease or condition or is expressed in the cells of the environment of a lesion associated with the disease or condition.
[96]
96. Polynucleotide, according to any of claims 63 to 95, characterized by the fact that the disease or condition is a cancer.
[97]
97. Polynucleotide, according to any of claims 63 to 96, characterized by the fact that the disease or condition is a myeloma, leukemia or lymphoma.
[98]
98. Polynucleotide, according to any of claims 63 to 97, characterized by the fact that the antigen is the B cell maturation antigen (BCMA), ROR1, carbonic anhydrase 9 (CAIX), teEGFR, Her2 / neu (receptor tyrosine kinase erbB2), L1- CAM, CD19, CD20, CD22, mesothelin, CEA and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-9), epithelial glycoprotein 40 (EPG-40), EPHa 2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vII, folate-binding protein (FBP), FCRL5, FOCRH5, fetal acetylcholine receptor, GD2, GD3, group D receptor member 5 coupled to protein G class C (GPRC5D), HMW-MAA, IL-92R-alpha, IL-13R-alpha2, receptor domain kinase insertion (kdr), leptin
ve cover, Lewis Y, L1 cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE) -A1, MAGE-A3, MAGE-AS6, Preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, I1L-13 (IL-13Ra2) alpha 2 receptor, CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7 / 8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5174, fetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testis antigen, small screen, Murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1I, TAG72, VEGF-R 2, carcinoembryonic antigen (CEA), Her2 / neu , estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD 2 (OGD2), CE7, Wilms' Tumor 1 (WT-1), a cyclin, cyclin A2, CCL -1, CD138, a pathogen specific antigen.
[99]
99. Polynucleotide according to claim 98, characterized by the fact that the antigen is a B cell maturation antigen (BCMA).
[100]
100. Polynucleotide, according to any of claims 63 to 99, characterized by the fact that the encoded antigen-binding domain is a fragment to the antibody that comprises a variable region of heavy chain (Vx) and a variable variable chain (V.).
[101]
101. Polynucleotide according to claim 100, characterized by the fact that: the Vu region is or comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the Vx region amino acid sequence mentioned in any of SEQ ID NOs: 617,115, 256, 519 or 609; and the V. region is or comprises an amino acid sequence
which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the region VI amino acid sequence mentioned in any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[102]
102. Polynucleotide according to claim 100 or 101, characterized by the fact that: the Vu region and the Vi regions comprise the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively, or an amino acid sequence which has at least 90% identity for SEQ ID NOS: 617 and 618, respectively; the Vx region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 256 and 267, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 256 and 267, respectively ; the Vx region and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 519 and 535, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 519 and 535, respectively ; the V region "and the V regions comprise the amino acid sequence mentioned in SEQ ID NOs: 115 and 536, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 115 and 536, respectively; or the V "region and the V. regions comprise the amino acid sequence mentioned in SEQ ID NOs: 609 and 610, respectively, or an amino acid sequence that has at least 90% identity to SEQ ID NOS: 609 and 610, respectively.
[103]
103. Polynucleotide according to any one of claims 100 to 102, characterized by the fact that the Vu region and the V region comprise the amino acid sequence mentioned in SEQ ID NOs: 617 and 618, respectively, or one string of friends
noacids that have at least 90% identity to SEQ ID NOS: 617 and 618, respectively.
[104]
104. Polynucleotide according to any one of claims 100 to 102, characterized by the fact that: the Vu region comprises a heavy chain complementarity determination region 1 (CDR-H1), a region for determining complementarity of heavy chain 2 (CDR-H2) and heavy chain 3 complementarity determining region (CDR-H3) contained within the amino acid sequence of region Vx selected from any of SEQ ID NOs: 617, 115, 256, 519 or 609; and the V. region comprises a light chain complementarity determination region 1 (CDR-L1), a light chain complementarity determination region 2 (CDR-L2) and a chain complementarity determination region take 3 (CDR-L3) contained within the amino acid sequence of region V. selected from any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[105]
105. Polynucleotide, according to any one of claims 100 to 104, characterized by the fact that: the Vx region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the mentioned amino acid sequence of the Vx region in SEQ ID NO: 617; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. mentioned in SEQ ID NO: 618.
[106]
106. Polynucleotide, according to any of claims 100 to 102 and 104, characterized by the fact that: the Vs region comprises (a) a CDR-H1 that comprises the selected amino acid sequence of any of SEQ ID NOs : 1, 2, 507 or 593; (b) a CDR-H2 comprising the selected amino acid sequence from any of the SEQ IDs
NOs: 4, 5, 513 or 594; and (c) a CDR-H3 comprising the selected amino acid sequence from any of SEQ ID NOs: 7, 10, 157, 517 or 595; and the V. region comprises (a) a CDR-L1 comprising the selected amino acid sequence from any of SEQ ID NOs: 33, 178, 380, 589 or 601; (b) a CDR-L2 comprising the selected amino acid sequence from any of SEQ ID NOs: 43, 183, 400, 590 or 602; and (c) a CDR-L3 comprising the selected amino acid sequence from any of SEQ ID NOs: 194, 416, 421, 591 or 603.
[107]
107. Polynucleotide, according to any of claims 100 to 102, 104 and 106, characterized by the fact that: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 2.5 and 157, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 178, 183 and 194, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 1,4 and 7, respectively, and the Vi region comprises a CDR-L1, CDR-L2 and CDR- L3 comprising the amino acid sequence of SEQ ID NOS: 380, 400 and 416, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 2, 5 and 10, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 33, 43 and 421, respectively; the Vs region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 507, 513 and 517, respectively, and the V. region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 589, 590 and 591, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615 and 603, respectively.
[108]
108. Polynucleotide, according to any one of claims 100 to 107, characterized by the fact that a Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and V. The region comprises CDR-L1, CDR-L2 and CDR-L3 which comprises the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively.
[109]
109. Polynucleotide, according to any one of the
vindications 100 to 102, 104, 106 and 107, characterized by the fact that the Vn region is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 617, 115, 256, 519 or 609; and region V. is or comprises the amino acid sequence mentioned in any of SEQ ID NOs: 618, 267, 535, 536 or 610.
[110]
110. Polynucleotide, according to any one of claims 100 to 102, 104, 106, 107 and 109, characterized by the fact that: the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617; and the V region is or comprises the amino acid sequence mentioned in SEQ ID NO: 618 the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 256; and the Vi region is or comprises the amino acid sequence mentioned in SEQ ID NO: 267; the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 519; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 535; the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 115; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 536; or the Vu region is or comprises the amino acid sequence mentioned in SEQ ID NO: 609; and the V. region is or comprises the amino acid sequence mentioned in SEQ ID NO: 610.
[111]
111. Polynucleotide, according to any one of claims 100 to 110, characterized by the fact that: the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 593 , 594 and 595, respectively; or a V4 region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOs: 611, 612 and 613, respectively; and the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602 and 603, respectively; or the V. region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 614, 615 and 603, respectively.
[112]
112. Polynucleotide according to claim 110 or 111, characterized in that the Vx region is or comprises the amino acid sequence mentioned in SEQ ID NO: 617; and region V. is or comprises the amino acid sequence mentioned in SEQ ID NO: 618.
[113]
113. Polynucleotide, according to any one of claims 100 to 112, characterized by the fact that the fragment comprises a scFv.
[114]
114. Polynucleotide, according to any one of claims 100 to 113, characterized by the fact that, when the Vu region, and the V. region are joined by a flexible ligand.
[115]
115. Polynucleotide according to claim 114, characterized by the fact that scFv comprises a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 361).
[116]
116. Polynucleotide, according to any of claims 100 to 115, characterized by the fact that the Vn region is an amino-terminal to the V ..
[117]
117. Polynucleotide according to any of claims 100 to 116, characterized by the fact that the antigen-binding domain comprises the amino acid sequence selected from any of SEQ ID NOs: 478, 278, 559, 560 or 442 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for amino acid sequence selected from any of SEQ ID NOs: 478, 278, 559, 560 or 442.
[118]
118. Polynucleotide according to any one of claims 100 to 117, characterized by the fact that the antigen-binding domain comprises the amino acid sequence mentioned in SEQ ID NO: 478 or an amino acid sequence that has at least minus 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity for the amino acid sequence mentioned in SEQ ID NO: 478.
[119]
119. Polynucleotide according to any one of claims 100 to 118, characterized by the fact that the antigen binding domain comprises the amino acid sequence mentioned in SEQ ID NO: 478.
[120]
120. Polynucleotide according to any of claims 100 to 119, characterized by the fact that the nucleic acid encoding the antigen-binding domain comprises (a) the nucleotide sequence mentioned in any of SEQ ID NOS : 648, 352, 647,716 or 718; (b) a nucleotide sequence that has at least 90% sequence identity to any of SEQ ID NOS: 648, 352, 647, 716 or 718; or (c) a degenerate sequence of (a) or (b).
[121]
121. Polynucleotide according to any one of claims 100 to 120, characterized by the fact that the nucleic acid encoding the antigen-binding domain comprises the nucleotide sequence mentioned in any of SEQ ID NO: 460, 440, 715, 717 or 719.
[122]
122. Polynucleotide according to any one of claims 100 to 121, characterized by the fact that the nucleic acid encoding the antigen-binding domain comprises the nucleotide sequence mentioned in SEQ ID NO: 460.
[123]
123. Polynucleotide, according to any one of the
vindications 100 to 116, characterized by the fact that a V "y region is a carboxy-terminal to the V region.
[124]
124. Polynucleotide encoding a chimeric antigen receptor, characterized by the fact that it comprises a nucleic acid that encodes: (1) an extracellular antigen binding domain that specifically binds to human B cell maturation antigen (BCMA) , in which the extracellular antigen-binding domain comprises: (i) a variable heavy chain (Vx) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% sequence identity for the Va region amino acid sequence mentioned in SEQ ID NO: 617; and (ii) a variable light chain region (V.) comprising a amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the mentioned V region amino acid sequence in any of SEQ ID NO: 618; (2) a spacer that comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint; an I9G2 / 4 chimeric Cn2 region; and an IgG4 Cr3 region, optionally about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD37) chain and an intracellular signaling domain of a T cell costimulatory molecule.
[125]
125. Polynucleotide according to claim 124, characterized by the fact that: the Vi region comprises CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the Vu region mentioned in SEQ ID NO: 617 ; and the V. region comprises CDR-L1, CDR-L2 and CDR-L3 contained within the amino acid sequence of region V. mentioned in SEQ ID NO: 618; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or the Vx region comprises a CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and the V region comprises a CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615 and 603, respectively.
[126]
126. Polynucleotide encoding a chimeric antigen receptor, characterized by the fact that it comprises a nucleic acid that encodes:
(1) an extracellular antigen-binding domain that specifically binds to human B-cell maturation antigen (BCMA), in which the extracellular antigen-binding domain comprises:
a variable heavy region (Vs) comprising CDR-H1, CDR-H2 and CDR-H3 contained within the Vx region amino acid sequence mentioned in SEQ ID NO: 617; and a light variable region (VL) comprising CDR-L1, CDR-L2 and CDR-L3 contained within the V region amino acid sequence mentioned in SEQ ID NO: 618; or a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 593, 594 and 595, respectively, and a V. region comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively;
a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOS: 596, 597 and 595, respectively, and a V. region comprising CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively;
a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 that comprise the amino acid sequence of SEQ ID NOS: 598, 599 and 595, respectively, and a V. region comprising CDR-L1, CDR-L2 and CDR -L3 comprising the amino acid sequence of SEQ ID NOS: 601, 602 and 603, respectively; or a V4 region comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612 and 613, respectively, and a V region comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615 and 603, respectively;
(2) a spacer that comprises a chimeric IgG4 / 2 joint or a modified IgG4 joint; an Ig9G2 / 4 chimeric CH2 region; and a CH3 region of IgG4, optionally about 228 amino acids in length; or a spacer mentioned in SEQ ID NO: 649; (3) a transmembrane domain, optionally a transmembrane domain of a human CD28; and (4) an intracellular signaling region that comprises a cytoplasmic signaling domain of a CD3-zeta chain (CD3C) and an intracellular signaling domain of a T cell costimulatory molecule.
[127]
127. Polynucleotide according to any of claims 124 to 126, characterized by the fact that the extracellular antigen binding domain comprises the amino acid sequence of region Vx mentioned in SEQ ID NO: 617, and the sequence of amino acids of region V. mentioned in SEQ ID NO: 618.
[128]
128. Polynucleotide, according to any one of claims 63 to 123, characterized by the fact that the intracellular signaling region comprises an activation cytoplasmic signaling domain.
[129]
129. Polynucleotide according to claim 128, characterized by the fact that the activation cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, it is a component of the T cell receptor (TCR) and / or it includes an immunoreceptor tyrosine-based activation motif (ITAM).
[130]
130. Polynucleotide according to claim 128 or 129, characterized in that the activation cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a CD3-zeta (CD36) chain or a variant function-
onal or signaling portion thereof.
[131]
131. Polynucleotide, according to any of claims 128 to 130, characterized by the fact that the cytoplasmic domain of activation is human or is a human protein.
[132]
132. Polynucleotide according to any one of claims 124 to 127, 130 and 131, characterized by the fact that the cytoplasmic signaling domain is or comprises the sequence mentioned in SEQ ID NO: 628 or an amino acid sequence that has at least 90% sequence identity of SEQ ID NO:
628.
[133]
133. Polynucleotide according to any of claims 124 to 127 and 130 to 132, characterized by the fact that the nucleic acid encoding the cytoplasmic signaling domain is or comprises the sequence mentioned in SEQ ID NO: 627 or is a codon-optimized sequence and / or its degenerate sequence.
[134]
134. Polynucleotide according to any of claims 124 to 127 and 130 to 133, characterized by the fact that the nucleic acid encoding the cytoplasmic signaling domain is or comprises the sequence mentioned in SEQ ID NO: 652.
[135]
135. Polynucleotide, according to any one of claims 128 to 134, characterized by the fact that the intracellular signaling region also comprises a co-stimulating signaling region.
[136]
136. Polynucleotide according to claim 135, characterized in that the co-stimulatory signaling region comprises an intracellular signaling domain of a T-cell co-stimulating molecule or a signaling portion thereof.
[137]
137. Polynucleotide according to claims 124 to 127, 135 and 136, characterized by the fact that the signaling region
Co-stimulatory training comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof.
[138]
138. Polynucleotide, according to any of claims 124 to 127 and 135 to 137, characterized by the fact that the co-stimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
[139]
139. Polynucleotide, according to any of claims 124 to 127 and 135 to 138, characterized by the fact that the co-stimulating signal region is human or is a human protein.
[140]
140. Polynucleotide according to any one of claims 124 to 127 and 135 to 139, characterized by the fact that the co-stimulating signal region is or comprises the sequence mentioned in SEQ ID NO: 626 or an amino acid sequence that exhibits at least 90% sequence identity for the sequence mentioned in SEQ ID NO: 626.
[141]
141. Polynucleotide according to any of claims 124 to 127 and 135 to 140, characterized by the fact that the nucleic acid encoding the co-stimulatory region is or comprises the sequence mentioned in SEQ ID NO: 625 or is a sequence codon-optimized and / or a degenerate sequence.
[142]
142. Polynucleotide according to any of claims 124 to 127 and 135 to 141, characterized by the fact that the nucleic acid encoding the co-stimulatory signaling region comprises the sequence mentioned in SEQ ID NO: 681.
[143]
143. Polynucleotide according to any of claims 63 to 139, characterized by the fact that the intracellular signaling region comprises the sequence mentioned in SEQ ID NO: 628 or an amino acid sequence that is at least 90% ,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for SEQ ID NO: 628, and the sequence mentioned in SEQ ID NO: 626 or a sequence of amino acids that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the sequence mentioned in SEQ ID NO:
626.
[144]
144. Polynucleotide according to any one of claims 63 to 139 and 143, characterized by the fact that the intracellular signaling region is or comprises the sequences mentioned in SEQ ID NO: 628 and SEQ ID NO: 626.
[145]
145. Polynucleotide, according to any one of claims 124 to 127, 135 to 137, 139 and 144, characterized by the fact that the co-stimulatory signaling region comprises an intracellular signaling domain of CD28.
[146]
146. Polynucleotide according to any of claims 63 to 139 and 145, characterized by the fact that the intracellular signaling region comprises the sequence mentioned in SEQ ID NO: 628 or an amino acid sequence that is at least 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity for SEQ ID NO: 628, and the sequence mentioned in SEQ ID NO: 680 or an amino acid sequence that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the sequence mentioned in SEQ ID NO : 680.
[147]
147. Polynucleotide, according to any of claims 63 to 139, 145 and 146, characterized by the fact that the intracellular signaling region is or comprises the sequences mentioned in SEQ ID NO: 628 and SEQ ID NO: 680.
[148]
148. Polynucleotide, according to any of claims 135 to 147, characterized by the fact that the region of
Co-stimulatory signaling is between the transmembrane domain and the intracellular signaling region.
[149]
149. Polynucleotide according to any of claims 63 to 148, characterized by the fact that the transmembrane domain is or comprises a transmembrane domain of CD4, CD28 or CD8.
[150]
150. Polynucleotide according to claim 149, characterized in that the transmembrane domain is or comprises a transmembrane domain of a CD28.
[151]
151. Polynucleotide, according to any of claims 63 to 150, characterized by the fact that the transmembrane domain is human or is a human protein.
[152]
152. Polynucleotide according to any of claims 63 to 151, characterized by the fact that the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 624 or an amino acid sequence that exhibits at least 90% of sequence identity for SEQ ID NO: 624.
[153]
153. Polynucleotide according to any of claims 63 to 152, characterized by the fact that the nucleic acid encoding the transmembrane domain is or comprises the sequence mentioned in SEQ ID NO: 623 or is an optimized sequence - used by codon and / or its degenerate sequence.
[154]
154. Polynucleotide according to claim 153, characterized in that the nucleic acid encoding the transmembrane domain comprises the sequence mentioned in SEQ ID NO: 688.
[155]
155. Polynucleotide, according to any of claims 63 to 154, characterized by the fact that the encoded chimeric antigen receptor comprises its N to C terminal in order: the antigen-binding domain, the spacer, the transmembrane domain, and the intracellular signaling region.
[156]
156. Polynucleotide, according to any of claims 63 to 155, characterized by the fact that the polynucleotide further encodes a truncated receptor.
[157]
157. Polynucleotide according to any of claims 63 to 156, characterized by the fact that the binding of the binding domain to the encoded antigen and / or the encoded chimeric antigen receptor or a measure indicative of function or activity of the chimeric antigen receptor encoded after exposure to cells expressing surface BCMA, is not reduced or blocked or is not substantially reduced or blocked in the presence of a soluble or spillable form of BCMA.
[158]
158. Polynucleotide according to claim 157, characterized by the fact that the concentration or quantity of the soluble or spillable form of BCMA corresponds to a concentration or quantity present in the serum or blood or plasma of the individual or a patient with multiple myeloma or on average in a patient population for the disease or disorder, or in a concentration or amount of soluble or spillable BCMA in which the binding or measure is reduced or blocked, or is substantially reduced or blocked, for cells that express a recombinant anti-BCMA reference receptor, optionally a reference anti-BCMA CAR, in the same assay.
[159]
159. Polynucleotide according to any of claims 63 to 158, characterized by the fact that the polynucleotide comprises the sequence mentioned in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the sequence mentioned in which- either SEQ ID NOS: 751-756, and the encoded receptor maintains the function of binding to BCMA, and the heterogeneity of RNA is reduced.
[160]
160. Polynucleotide according to any of claims 63 to 159, characterized by the fact that the polynucleotide comprises the sequence mentioned in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for the sequence mentioned in which- either SEQ ID NOS: 755 and 756, and the encoded receptor maintains the function of binding to BCMA, and the heterogeneity of RNA is reduced.
[161]
161. Polynucleotide according to any of claims 63 to 160, characterized by the fact that the polynucleotide comprises the sequence mentioned in SEQ ID NOs: 755 or a sequence that exhibits at least or at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the encoded receiver maintains the function of binding to BCMA and maintains the heterogeneity of the Reduced RNA.
[162]
162. Polynucleotide, according to any of claims 63 to 161, characterized by the fact that the polynucleotide comprises the sequence mentioned in SEQ ID NOs: 755, and the encoded receptor maintains the function of binding to BCMA and keeps the RNA heterogeneity reduced.
[163]
163. Vector, characterized by the fact that it comprises the polynucleotide as defined in any one of claims 63 to 162.
[164]
164. Vector, according to claim 163, characterized by the fact that the vector is a viral vector.
[165]
165. Vector, according to claim 164, characterized by the fact that the viral vector is a retroviral vector.
[166]
166. Vector according to claim 164 or 165, characterized by the fact that the viral vector is a lentiviral vector.
[167]
167. Chimeric antigen receptor, characterized by the fact that it is encoded by the polynucleotide as defined in any one of claims 63 to 162.
[168]
168. Modified cell, characterized by the fact that it comprises the chimeric antigen receptor as defined in any one of claims 1 to 62 and 167.
[169]
169. Modified cell, characterized by the fact that it comprises the polynucleotide as defined in any of claims 63 to 162 or the vector as defined in any of claims 163 to 166.
[170]
170. Modified cell according to claim 168 or 169, characterized by the fact that the cell is an immune cell.
[171]
171. Modified cell according to any one of claims 168 to 170, characterized by the fact that the immune cell is a primary cell obtained from an individual.
[172]
172. Modified cell according to claim 170 or 171, characterized by the fact that the immune cell is an NK cell or a T cell.
[173]
173. Modified cell according to any of claims 170 to 172, characterized by the fact that the immune cell is a T cell, and the T cell is a CD4 + and / or CD8 + T cell.
[174]
174. Modified cell according to any one of claims 168 to 173, characterized by the fact that the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mMRNA), which exhibits at least 70%, 75%, 80%, 85%, 90% or 95% RNA homogeneity.
[175]
175. Modified cell according to any one of claims 168 to 174, characterized by the fact that the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mMRNA), which exhibits reduced heterogeneity compared to mMRNA heterogeneity transcribed in a cell encoding a reference chimeric antigen receptor, said reference chimeric antigen receptor that comprises the same amino acid sequence as the chimeric antigen receptor, however, encoded by a different polynucleotide sequence comprising one or more nucleotide differences in the polynucleotide encoding the CARs and / or in which the reference chimeric antigen receptor is encoded by a polynucleotide comprising one or more binding donor sites and / or one or more receptor binding sites on the nucleic acid encoding the spacer.
[176]
176. Modified cell according to claim 175, characterized by the fact that the RNA heterogeneity is reduced by more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50 % or more.
[177]
177. Modified cell according to claim 175 or 176, characterized by the fact that the polynucleotide encoding the reference CAR comprises the transcribed RNA encoding the reference CAR, optionally the messenger RNA (mMRNA), which exhibits more than or more than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity.
[178]
178. Modified cell according to any one of claims 174 to 177, characterized by the fact that RNA homogeneity and / or heterogeneity is / are determined by agarose gel electrophoresis, capillary electrophoresis based on chip, analytical ultracentrifugation, field flow fractionation or liquid chromatography.
[179]
179. Modified cell according to any of claims 168 to 178, characterized by the fact that, among a plurality of modified cells, less than or less than about
10%, 9%, 8%, 7%, 5%, 4%, 3%, 2% or 1% of cells in plurality comprise a chimeric antigen receptor that exhibits tonic signaling and / or activity or signaling independent of antigen.
[180]
180. Composition, characterized by the fact that it comprises the chimeric antigen receptor as defined in any of any of claims 1 to 62 and 167, the polynucleotide as defined in any of claims 63 to 162, or the vector as defined in any one of claims 163 to 166.
[181]
181. Composition, characterized by the fact that it comprises the modified cell as defined in any of claims 168 to 179.
[182]
182. Composition according to claim 181, characterized in that the composition comprises CD4 + and CD8 + T cells, and the ratio of CD4 + T cells to CD8 + is about 1: 3 to 3: 1.
[183]
183. Composition according to any one of claims 180 to 182, characterized by the fact that it also comprises a pharmaceutically acceptable excipient.
[184]
184. Method of treatment, characterized in that it comprises the administration of the modified cell as defined in any of claims 168 to 179 or the composition as defined in any of claims 180 to 183 to an individual with a disease or disorder.
[185]
185. Method according to claim 184, characterized in that the method comprises administering a dose of the modified cells or a composition comprising a dose of the modified cells.
[186]
186. Use of the modified cell as defined in any of claims 168 to 179 or the composition as defined in any of claims 180 to 183, characterized by the fact that it is for the manufacture of a medicament for the treatment of a disease or disorder .
[187]
187. Use of the modified cells as defined in any of claims 168 to 179 or the composition as defined in any of claims 180 to 183, characterized by the fact that it is for the treatment of a disease or disorder.
[188]
188. Use according to claim 186 or 187, characterized by the fact that the modified cells or composition are for use in a treatment regime, wherein the treatment regime comprises administering a dose of the modified cells or a composition comprising a dose of the modified cells.
[189]
189. Method according to claim 184 or 185, or use according to any of claims 186 to 188, characterized by the fact that the disease or disorder is associated with the expression of the B cell maturation antigen ( BCMA), optionally a B cell related disorder.
[190]
190. Method or use according to any of claims 184 to 189, characterized in that the disease or disorder associated with BCMA is an autoimmune disease or disorder.
[191]
191. Method or use according to any of claims 185 to 190, characterized by the fact that the disease or disorder associated with BCMA is a cancer.
[192]
192. Method according to claim 191, characterized by the fact that cancer is a BCMA-expressing cancer.
[193]
193. Method according to claim 191 or 192, characterized by the fact that cancer is a B cell malignancy.
[194]
194. Method, or use according to any of claims 191 to 193, characterized by the fact that cancer is a lymphoma, leukemia or malignancy of plasma cells.
[195]
195. Method according to claim 194, characterized by the fact that the cancer is a lymphoma, and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, lymphoma follicular, non-cleaved small cell lymphoma, mucosal lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocyte B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, mixed cell lymphoma diffuse, angiocentric pulmonary B-cell lymphoma, small lymphocytic lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmocytic lymphoma) or liner cell lymphoma (MCL).
[196]
196. Method according to claim 195, characterized by the fact that cancer is leukemia, and leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL).
[197]
197. Method according to claim 194, characterized by the fact that cancer is a malignancy of plasma cells, and the malignancy of plasma cells is multiple myeloma (MM) or plasmacytoma.
[198]
198. Method or use according to any of claims 191 to 194 and 197, characterized by the fact that cancer is multiple myeloma (MM).
[199]
199. Method according to any of claims 185 and 188 to 198, characterized in that the dose of modified T cells comprises between or about 1 x 107 T cells of CAR expression and in or about 2 x 10th T cells of CAR expression or between or at or about.
[200]
200. Method or use according to any one of claims 185 and 188 to 199, characterized in that the dose of modified T cells comprises between or about 2.5 x 107 T cells of CAR expression and in or about 1.2 x 10º CAR expression T cells, enter in or about 5.0 x 107 CAR expression T cells and in or about 4.5 x 10º CAR expression T cells or in or about 1.5 x 10º CAR expression T cells and about 3.0 x 10º CAR expression T cells.
[201]
201. Method, or use according to any of claims 185 and 188 to 200, characterized in that the dose of modified T cells comprises at or about 2.5 x 10 ”, at or about 5.0 x 107, at or about 1.5 x 108, at or about 1.5 x 108, at or about 3.0 x 108, at or about 4.5 x 108, at or about 8.0 x 10º or in or about 1.2 x 10º T cells of CAR expression.
[202]
202. Method, or use according to any one of claims 185 and 188 to 201, characterized in that the dose of modified T cells comprises at or about 5.0 x 107, at or about 1.5 x 108, at or about 1.5 x 108º, in or about 3.0 x 10º or in or about 4.5 x 10º T cells of CAR expression.
[203]
203. Method, or use according to any one of claims 185 and 188 to 202, characterized in that the dose of modified T cells comprises a combination of CD4 + T cells and CD8 + T cells, in a T cell ratio of expression of CD4 + CAR for T cells of expression of CAR CD8 + and / or of CD4 + T cells for CD8 + T cells, which is either approximately 1: 1 or approximately 1: 3 to approximately 3: 1.
[204]
204. Method or use according to any of claims 185 and 188 to 203, characterized by the fact that less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6 %, 5%, 4%, 3%, 2% or 1% of CAR expression T cells in the modified T cell dose express an apoptosis marker, optionally Annexin V or active Caspase 3.
[205]
205. Method or use according to any of claims 185 and 188 to 204, characterized by the fact that less than 5%, 4%, 3%, 2% or 1% of the CAR expression T cells in the if modified T cells express Annexin V or active Caspase 3.
[206]
206. Method or use according to any one of claims 184, 185 and 188 to 205, characterized by the fact that, prior to administration, the individual received a lymphatic drainage therapy comprising the administration of fludarabine in or about 20-40 mg / m in the individual's body surface area, optionally at or about 30 mg / m , daily, for 2-4 days, and / or cyclophosphamide at or about 200-400 mg / m on the individual's body surface area, optionally at or about 300 mg / m , daily, for 2-4 days.
[207]
207. Method or use according to any one of claims 184, 185 and 188 to 206, characterized by the fact that the individual has received a lympho-exhaustion therapy that comprises the administration of fludarabine at or about 30 mg / m on the individual's body surface, daily and cyclophosphamide at or about 300 mg / m on the individual's body surface, daily, for 3 days.
[208]
208. Method or use according to any one of claims 184, 185 and 188 to 207, characterized in that, on or before the administration of the cell dose, the individual received three or more previous therapies for the disease or disorder , optionally four or more previous therapies, optionally selected from: autologous stem cell transplantation (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody.
[209]
209. Method or use according to any of claims 208, characterized by the fact that the immunomodulating agent
The controller is selected from thalidomide, lenalidomide, and pomalido- mide.
[210]
210. Method according to claim 208 or 209, characterized by the fact that the proteasome inhibitor is selected from bortezomib, carfilzomib and ixazomib.
[211]
211. Method or use according to any one of claims 208 to 210, characterized in that the anti-CD38 antibody is or comprises daratumumab.
[212]
212. Method or use according to any one of claims 184, 185 and 188 to 211, characterized by the fact that, at the time of administration of the cell dose, and / or at the time of lymphoblastic chemotherapy or lymphodetective leukopheresis, the individual was not active or had a history of plasma cell leukemia (PCL).
[213]
213. Method or use according to any of claims 184, 185 and 188 to 212, characterized by the fact that at the time of cell dose administration, the individual developed secondary plasma cell leukemia (PCL).
[214]
214. Method or use according to any of claims 184, 185 and 188 to 213, characterized by the fact that, at the time of administration, the individual: relapsed or was refractory after at least 3 or at least 4 previous therapies for multiple myeloma; is an adult individual or is 25 or 35 years of age or older; has a time of diagnosis of multiple myeloma of approximately 4 years or between 2 and 15 or 2 and 12 years; received about 10 or between 3 and 15 or between 4 and 15 previous regimens for multiple myeloma; was refractory or did not respond to bortezomib,
be, lenalidomide, pomalidomide and / or an anti-CD38 monoclonal antibody; had previous autologous stem cell transplantation or had no previous autologous stem cell transplantation; and / or has high risk cytogenetics for IMWG.
[215]
215. Method or use according to any of claims 184 to 214, characterized by the fact that the method is capable of achieving a specified answer or result, optionally at a designated point in time after the start of administration , in at least one or at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, 60%, 70%, 80%, 90% or 95% of individuals in a cohort of individuals with the individual's disease or disorder, optionally, where the cohort of individuals has at least the same number of previous therapies, prognosis or prognostic factor, subtype, secondary involvement or other specific aspects or characteristics of the patient, such as individual treated by the method, in which: the response is selected from the group consisting of objective response (OR), complete response (CR), strict complete response (sCR), very good partial response (VGPR), partial response (PR ) and minimum response (MR); the answer or result is or comprises an OR the answer or result is or comprises a CR.
[216]
216. Method or use according to claim 215, characterized by the fact that the answer or result is an OR and is achieved by at least 40%, at least 50%, at least 60%, by at least 70% or at least 80% of the individuals in the cohort.
[217]
217. Method according to claim 215, characterized by the fact that the answer or result is a CR or sCR and is achieved in at least 20%, 30% or 40% of the individuals in the cohort.
[218]
218. Method or use according to any of claims 215 to 217, characterized by the fact that the cell dose is less than 1.5 x 108 cells or less than 1.5 x 10% 8 CAR + cells T or less than 3 x 10º8 CAR + T cells or less than 4.5 x 108 CAR + T cells.
[219]
219. Method or use according to any of claims 215 to 217, characterized by the fact that the cell dose is in or about 1.5 x 1068 cells or less than 1.5 x 1058 cells las CAR + T.
[220]
220. Method or use according to any of claims 215 to 219, characterized by the fact that the cell dose is in or about 5 x 107 cells or CAR + T cells.
[221]
221. Method or use according to any of claims 215 to 219, characterized by the fact that the cell dose is in or about 1.5 x 10 th cells or CAR + T cells.
[222]
222. Method or use according to any of claims 215 to 219, characterized by the fact that the cell dose is in or about 3 x 10th cells or CAR + T cells.
[223]
223. Method or use according to any of claims 215 to 219, characterized by the fact that the cell dose is in or about 4.5 x 10 th cells or CAR + T cells.
[224]
224. Method or use according to any one of claims 215 to 223, characterized by the fact that the answer or result comprises or still comprises the absence of grade 3 or higher neurotoxicity, or grade 4 or higher, at absence of grade 3 or higher or grade 4 or higher, of the cytokine release syndrome.
[225]
225. Method or use according to any one of claims 215 to 224, characterized in that the dose of modified T cells comprises at or about 5.0 x 10 ”, at or about 1.5 x 108, in or about 3.0 x 10 ° or in or about 3.0 x 108 or in or about 4.5 x 10 T cells of CAR expression.
[226]
226. Method or use according to any of claims 215 to 225, characterized by the fact that the dose of the modified T cells comprises in or about 5.0 x 10º T cells of CAR expression.
[227]
227. Method or use according to any of claims 215 to 225, characterized in that the dose of the modified T cells comprises in or about 1.5 x 10 T cells of CAR expression.
[228]
228. Method or use according to any one of claims 215 to 225, characterized in that the dose of the modified T cells comprises in or about 3 x 108 T cells of CAR expression.
[229]
229. Method or use according to any of claims 215 to 225, characterized in that the dose of the modified T cells comprises or about 4.5 x 108 T cells of CAR expression.
[230]
230. Cell according to any of claims 168 to 179, or composition according to any of claims 180 to 183, characterized by the fact that the cell or composition, after administration in a dose of CAR + cells , is able to optionally achieve, at a designated time after the start of administration, a specified response or result in at least one of, or at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of individuals within a cohort of individuals or assessable individuals thereof, where the cohort of individuals is a cohort with multiple myeloma.
[231]
231. Cell or composition according to claim 230, characterized by the fact that obtaining the answer or result is at the designated time after the start of administration, which is at least 1, 2, 3, 6, 9 or 12 months after the start.
[232]
232. Cell or composition according to claim 231, characterized by the fact that obtaining the response or result is at the designated time after the start of administration, which is at least 1 or 2 or 3 months after said start .
[233]
233. Cell or composition, according to claim 230, characterized by the fact that: the cohort of individuals is that of individuals with relapsed or refractory multiple myeloma; the cohort of individuals is individuals with relapsed or refractory multiple myeloma who have been administered, and relapsed or refractory after at least three previous therapies for multiple myeloma, said previous therapies optionally including an immunomodulatory agent; a proteasome inhibitor; and / or an anti-CD38 antibody; the cohort of individuals is individuals with relapsed or refractory multiple myeloma who have been administered and relapsed or refractory after at least 3 previous therapies for multiple myeloma, said previous therapies optionally including an immunomodulatory agent; a proteasome inhibitor; and / or an anti-CD38 antibody and / or an autologous stem cell transplant; and / or the cohort of individuals are individuals who do not have active plasma cell leukemia (PCL) or without a history of PCL at the time of said administration; the cohort of individuals is individuals who developed secondary plasma cell leukemia (PCL) prior to administration of the cells the cohort of individuals is or includes individuals with relapsed or refractory multiple myeloma being administered and relapsed or refractory after at least 4 or an average of at least 10 previous therapies for multiple myeloma; the cohort of individuals consists of or includes adult individuals; the cohort of individuals has a median time since the diagnosis of 4 years and / or a diagnostic time range of 2 to 12 years; the cohort of individuals received an average of 10 previous regimens or between 3 and 15 or 4 and 15 previous therapies for multiple myeloma; the cohort of individuals includes individuals refractory to borzozomib, carfilzomib, lenalidomide, pomalidomide and a monoclonal anti-CD38 antibody; the cohort of individuals includes individuals who have had previous autologous stem cell transplantation; and / or the cohort of individuals includes individuals with high risk cytogenetics for IMWG.
[234]
234. Cell or composition according to claim 233, characterized by the fact that the immunomodulatory agent is selected from thalidomide, lenalidomide, and pomalidomide, the proteasome inhibitor is selected from bortezomib, carfilzomib and ixa-zomib and / or the anti-CD38 antibody is or comprises daratumumab.
[235]
235. Cell or composition according to any one of claims 230 to 234, characterized by the fact that the answer or result is selected from the group consisting of objective answer (OR), complete answer (CR), complete answer rigorous (sSCR), very good partial response (VGPR), partial response (PR) and minimal response (MR), optionally based on the uniform response criteria of the International Myeloma Working
Group (IMWG); the answer or result is or comprises an OR, optionally based on the uniform response criteria of the International Myeloma Working Group (IMWG); or the answer or result is or comprises a CR, optionally based on the uniform response criteria of the International Myeloma Working Group (IMWG).
[236]
236. Cell or composition according to any one of claims 230 to 235, characterized by the fact that the answer or result is or comprises an OR.
[237]
237. Cell or composition according to any one of claims 230 to 236, characterized in that the dose is capable of achieving the response or the result in at least 40%, at least 50%, at least 60%, at least at least 70% or at least 80% of the individuals in the cohort.
[238]
238. Cell or composition according to any one of claims 230 to 235, characterized by the fact that the answer or result is or comprises a CR or sCR.
[239]
239. Cell or composition according to any one of claims 230 to 238, characterized by the fact that the dose is capable of achieving the response or result in at least 20%, 30% or 40% of the individuals in the cohort.
[240]
240. Cell or composition according to any one of claims 230 to 239, characterized in that the dose capable of achieving said response or result is less than 1.5 x 108 cells the dose capable of achieving said response or result is less than 1.5 x 1098 CAR + T cells.
[241]
241. Cell or composition according to any one of claims 230 to 240, characterized in that the dose capable of achieving said response or result is less than 1.5 x 108 cells the dose capable of achieving said response or result is less than 1.5 x 1068 CAR + T cells; the dose capable of achieving that response or result is less than 3 x 108 CAR + T cells; or the dose capable of achieving that response or result is less than 4.5 x 1068 CAR + T cells.
[242]
242. Cell or composition according to any one of claims 230 to 241, characterized in that the dose capable of achieving said response or result is less than 1 x 108 cells the dose capable of achieving said response or result is less than 1 x 108 CAR + T cells.
[243]
243. Cell or composition according to any one of claims 230 to 242, characterized in that the dose capable of achieving said response or result is about 5 x 107 cells or in or about 5 x 107 CAR + cells T.
[244]
244. Cell or composition according to any one of claims 230 to 243, characterized in that the dose capable of achieving said response or result is in or about 1.5 x 1098 CAR + T cells or cells.
[245]
245. Cell or composition according to any one of claims 230 to 244, characterized in that the dose capable of achieving said response or result is in or about 3 x 1058 CAR + T cells or cells.
[246]
246. Cell or composition according to any one of claims 230 to 245, characterized in that the dose capable of achieving said response or result is in or about 4.5 x 108 cells or CAR + T cells.
[247]
247. Cell or composition according to any one of claims 230 to 246, characterized by the fact that the answer or result comprises or further comprises the absence of grade 3 or higher or grade 4 or higher neurotoxicity absence of grade 3 or higher or grade 4 or higher, of the cytokine release syndrome.
[248]
248. Method for determining the heterogeneity of a transgene nucleic acid transcribed from a transgene, the method characterized by the fact that it comprises: a) amplifying a transcribed nucleic acid using at least one pair of primers in 5 'and 3, in that at least one pair comprises a 5 'primer that is complementary to a nucleic acid sequence within the 5' (5 'UTR) untranslated region of the transcribed nucleic acid and a 3' primer that is complementary to a nucleic acid sequence within the 3 '(3' UTR) untranslated region of the transcribed nucleic acid to generate one or more amplified products; and b) detecting the amplified products, in which the presence of two or more amplified products from at least one primer pair in 5 'and 3' indicates heterogeneity in the amplified products.
[249]
249. Method according to claim 248, characterized by the fact that the differences in b) are different lengths from the amplified transcripts.
[250]
250. Method according to claim 248, characterized by the fact that the differences in b) are differences in the chromatographic profiles of the amplified transcripts.
[251]
251. Method according to any one of claims 248 to 250, characterized by the fact that differences in amplified products are determined by agar-gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation.
field flow fractionation or chromatography.
[252]
252. Method according to any one of claims 248 to 251, characterized in that the 5 'primer is specific for the transcribed sequence of the transcribed nucleic acid promoter region.
[253]
253. Method according to any one of claims 248 to 252, characterized in that the transcribed nucleic acid is amplified using a specific 3 'primer for a sequence within the polynucleus amino acid coding sequence - cleotide and / or the 3 'untranslated region of the transcribed pre-mRNA.
[254]
254. Method according to any one of claims 248 to 253, characterized in that the primer in 3 is specific for the polyadenylation sequence or enhancer region of the 3 'untranslated region of the transcribed pre-mRNA .
[255]
255. Method according to any one of claims 248 to 254, characterized by the fact that step a) is carried out by a single amplification reaction, using a single pair of 5 'and 3 initiators, comprising a 5 'primer that is complementary to a nucleic acid sequence within the 5' (5 'UTR) untranslated region of the transcribed nucleic acid and a 3' primer that is complementary to a nucleic acid sequence within the 3 '(3' RTU) untranslated region.
[256]
256. Method according to any one of claims 248 to 255, characterized in that step a) is carried out by parallel or subsequent amplification reactions using a first 5 'and 3' primer pair, a second 5 'and 3' primer pair and, optionally, 5 'and 3' primer pairs, where: the first 5 'and 3' primer pair contains a 5 'primer that is complementary to a sequence of nucleic acid within the 5 'UTR of the transcribed nucleic acid and a 3' primer that is complementary to a nucleic acid sequence within the 3 'UTR of the transcribed nucleic acid; the second 5 'and 3' primer pair contains a 5 'primer whose sequence is complementary to a portion of the translated nucleic acid transcription sequence and a 3' primer whose sequence is complementary to a nucleic acid sequence within the 3 'transcription RTU; and the optionally additional additional 5 'and 3' primer pairs each contain sequences complementary to the sequences within the translated region of the transcript.
[257]
257. Method according to claim 256, characterized by the fact that parallel or subsequent amplification reactions amplify overlapping portions of the transcription.
[258]
258. Method according to any one of claims 248 to 257, characterized in that the amplified products are predicted to be about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 kilobases, 4.5 kilobases, 5 kilobases, 5.5 kilobases, 6 kilobases, 7 kilobases or 8 kilobases in length.
[259]
259. Method according to any one of claims 248 to 258, characterized in that a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more binding sites.
[260]
260. Method according to claim 259, characterized in that the transgenic candidate's transcribed nucleic acid exhibits at least or at least about 5%, 10%, 15%, 20%, 25% , 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity after expression in a cell.
[261]
261. Method to reduce the heterogeneity of a transmission
creation of expressed transgene, characterized by the fact that it comprises: a) identifying a candidate for transgene for the removal of binding sites according to the method as defined in claim 259 or 260; b) identify one or more potential donor and / or binding receptors sites; and c) modifying the nucleic acid sequence at or near one or more binding donor sites and / or potential binding receptors identified in b), thereby generating a modified polynucleotide.
[262]
262. Method, according to claim 261, characterized by the fact that it also comprises: d) evaluating the transgenic candidacy for the removal of binding sites, as in step a).
[263]
263. Method according to claim 262, characterized by the fact that it further comprises e) repeating steps b) -d) until the heterogeneity of the transcription in step d) is reduced compared to the heterogeneity of the transcription, as determined in step a).
[264]
264. Method according to any one of claims 261 to 263, characterized in that the one or more potential donor and / or binding receptor sites exhibit a score of about or at least about 0, 7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.0 of a link event and / or is / are expected to be involved in a link event with a probability at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
[265]
265. Method according to any one of claims 261 to 264, characterized by the fact that the donor binding sites and the binding receptor sites are identified independently.
[266]
266. Method according to any of claims 261 to 265, characterized by the fact that the recipient site (s) and / or link donor (s) is / are site ( s) canonical, non-canonical (s) and / or cryptic linker (s) or donor (s).
[267]
267. Method according to any of claims 261 to 266, characterized by the fact that the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor.
[268]
268. The method of claim 267, characterized in that the CAR polypeptide comprises an antigen-binding domain comprising an antibody fragment, optionally a single chain antibody (scFv) fragment, comprising a variable heavy chain (Vx) and variable light chain (V.), a spacer, a transmembrane region and an intracellular signaling region.
[269]
269. Method according to claim 267 or 268, characterized in that the modified polynucleotide is not modified within the coding sequence for the antigen binding domain of the encoded CAR polypeptide.
[270]
270. Method according to any one of claims 261 to 269, characterized in that the encoded amino acid sequence of the transgene is unchanged after modification of the polylucleotide.
[271]
271. Method according to any of claims 261 to 270, characterized in that the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80% , 90% or 95% homogeneity after expression of the unmodified polynucleotide in a cell.
[272]
272. Method according to any one of claims 248 to 271, characterized by the fact that the cell is a human cell.
[273]
273. Method according to any of claims 248 to 272, characterized in that the cell is a T cell.
[274]
274. Method according to any one of claims 248 to 273, characterized by the fact that the method is a method implemented by a computer and in which one or more steps a) -c) take place in an electronic device comprising a or more processors and memory.
[275]
275. Computer system, characterized by the fact that it comprises a processor and memory, the memory comprising instructions operable to make the processor perform any one or more steps of the methods as defined in any one of claims 248 to 274 .

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同族专利:

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公开号 | 公开日

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WO2019090003A8|2019-07-11|

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IL274292D0|2020-06-30|

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US20190161553A1|2019-05-30|

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CO2020006441A2|2020-08-21|

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WO2019090003A1|2019-05-09|

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CN111902159A|2020-11-06|

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US11066475B2|2021-07-20|

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US20210324100A1|2021-10-21|

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CA3082010A1|2019-05-09|

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WO2019090003A9|2019-09-19|

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EP3703750A1|2020-09-09|

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MA50860A|2020-09-09|

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KR20200116077A|2020-10-08|

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SG11202003866QA|2020-05-28|

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TW201932482A|2019-08-16|

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CL2020001151A1|2020-10-23|

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AU2018360800A1|2020-05-14|

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JP2021501606A|2021-01-21|

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762580445P| true| 2017-11-01|2017-11-01|

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US201762580439P| true| 2017-11-01|2017-11-01|

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US62/580,439|2017-11-01|

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US62/580,445|2017-11-01|

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US201762582938P| true| 2017-11-07|2017-11-07|

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US201762582932P| true| 2017-11-07|2017-11-07|

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US62/582,932|2017-11-07|

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US62/582,938|2017-11-07|

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US201762596763P| true| 2017-12-08|2017-12-08|

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US201762596765P| true| 2017-12-08|2017-12-08|

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US62/596,765|2017-12-08|

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US201862614960P| true| 2018-01-08|2018-01-08|

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US201862614963P| true| 2018-01-08|2018-01-08|

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US201862665442P| true| 2018-05-01|2018-05-01|

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